Compositions and methods for reducing the transmissivity of illnesses

ABSTRACT

Methods for prophylactic and anti-transmissivity uses of an anti-microbial composition are disclosed. The methods comprise the step of administering to a mammal or a bird, an amount of a composition having a first ingredient obtainable from ginger; a second ingredient obtainable from green tea; an optional third ingredient obtainable from turmeric; and an acceptable carrier. When administered the composition is effective to reduce the incidence of contracting an illness or to prophylactically prevent transmission of an illness. Also disclosed are nasal and throat spray compositions for use in the methods of the invention.

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/359,889 filed on Feb. 6, 2003, currently pending; which, inturn, is a continuation-in-part of International Patent Application No.PCT/US02/24794, filed on Aug. 6, 2002, designating the United States ofAmerica and published in English; which, in turn, is acontinuation-in-part of U.S. patent application Ser. No. 10/122,991,filed on Apr. 15, 2002, now U.S. Pat. No. 6,596,313; which, in turn, isa continuation-in-part of U.S. patent application Ser. No. 09/923,090,filed on Aug. 6, 2001, now U.S. Pat. No. 6,592,896.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates the prophylactic use of a composition toreduce the incidence of contraction of illnesses caused by microbialorganisms. More particularly, the present invention relates to methodsfor treating, reducing or preventing one or more symptoms or adverseeffects of a microbial infection and to methods for reducing theinfectivity or transmission of microbial infections.

2. Description of the Related Technology

Viral pathogenesis is the method by which viruses produce disease in thehost. The pathogenesis of viruses centers on the mechanisms of viralinjury to discrete populations of cells in particular organs to producesigns and symptoms of disease in a particular host.

To initiate an infection the virus must gain entry to the host cell.Entry routes are dependent on the virus and include the skin, eyes,respiratory, GI and urogenital tracts as well as the circulatory system.Some viruses localize their tissue injury in close proximity to theirsite of entry, particularly the viruses that infect the upperrespiratory tract such as influenza, parainfluenza, rhinoviruses andcoronavirus. Once the viral particle has invaded the cell, viral codedproteins direct the cell to replicate the viral genome and produce viralspecific proteins. These proteins are assembled into complete virionsalong with the viral genome and released. In the case of envelopedviruses, the virions acquire a lipid membrane and will insert throughthis lipid membrane, viral specific glycoproteins. The enveloped virusfamilies include the Herpesviridae, Retroviridae, Orthomyxoviridae,Paramyxoviridae, Flaviviridae, Togaviridae and Coronaviridae. Therhinoviruses are members of the Picornaviridae, which are not enveloped.

Viruses have evolved a number of mechanisms to enter a host cell andinitiate infection. To fuse to the cell membrane, viruses have amembrane glycoprotein with membrane fusion activity. Many envelopedvirus induce a receptor-mediated endocytosis after binding to the cellsurface receptor, causing the cell to form an endosomal vesicle. Onceinside the vesicle, the virus particle undergoes the uncoating process.This insures that the optimal pH for the viral genome is maintained andthat the viral genome is protected from cellular nucleases.

Influenza viruses belong to the Orthomyxoviridae family of viruses andthey are enveloped viruses containing negative-stranded RNA genomes witheight segments. The viral RNA encodes 10 viral specific proteins.Initiation of the infective cycle requires the binding of the viralenvelop to the host cell-surface receptors, followed byreceptor-mediated endocytosis and the fusion of the viral and endosomalmembranes. The fusion process allows the release of the viral genomeinto the cytoplasm, where it can migrate to the nucleus where the viralgenome initiates viral transcription and replication. The proteinresponsible for influenza receptor binding and membrane fusion is thehemagglutinin protein (HA or H antigen). For most strains, the HAprotein is the most abundant glycoprotein on the surface of the virion.The HA protein is also the target for neutralizing antibodies. There arethree serotypes of influenza viruses: A, B and C. Serotypes A and Bcause the majority of clinical diseases. Influenza A occurs the mostfrequently, it is more virulent and it is responsible for the majorityof epidemics and pandemics. Influenza A can be further subtyped based onthe surface antigens HA and neuraminidase (N antigen) and the H and Nantigens are the major antigenic determinants. Strains are alsoclassified based on geographical location of the first isolate, serialnumber and year of isolation. Neuraminidase is an enzyme thatfacilitates the release of new viral particles from infected host cell.A third protein, M protein (matrix protein), is a membrane channelprotein and is known as M2 in the A strains and NB in B strains. Thesesurface viral membrane glycoproteins are the targets against which theimmune system reacts.

Influenza viral particles attach to epithelial cells in the upper andlower respiratory track, where they invade the cell, release theirgenome and subjugate the host cell replication machinery to reproduceviral proteins and nucleic acid. Mature viral particles are released bylysis of the host cell. The resulting breaches in the respiratoryepithelium results in an increase susceptibility to secondary infection.Influenza is transmitted primarily by respiratory secretions and thesesecretions are spread by coughing and sneezing. Influenza is also spreadby direct contact when hands contaminated with the virus come in directcontact with the nasal passages or the eye. The incubation period isfrom 1 to 4 days and infected persons are generally infectious a day ortwo before symptoms appear and can remain infectious for 5 days afterthe onset of illness. Children and the immunocompromised shed virus forlonger periods.

Influenza is prone to minor changes (i.e. point mutations) to one orboth of the major surface antigens during replication. These changes aredue in part to the lack of proofreading and error correction mechanismsin the virus transcriptional apparatus. The so-called antigenic drift isresponsible for the seasonal epidemics because it can enable the virusto infect persons with only partial immunity from a prior exposure tothe virus. Influenza A viruses are especially prone to antigenic drift.Major changes in the H and N antigens result in antigenic shift.Antigenic shift results in a new viral subtype and it can cause majorepidemics and pandemics due to minimal populational immunity.

Influenza has been established as a serious human affliction that cancause localized epidemics and global pandemics of acute respiratoryinfections. Each year the influenza virus is responsible for 20,000 to40,000 deaths and up to 300,000 hospitalization cases in the US. (Sandhaand Mossad, Influenza in the Older Adult. Indications for the Use ofVaccine and Antiviral Therapy, Geriatrics 56: 43-51, 2001, Oxford et al,In: Antigenic Variation, Ed. Craig & Scherf, Academic Press, London pp.53-83, 2003). In the pandemic of 1918 it is widely believed that anexcess of 40 million people died. Although children and younger adultsexperience more cases of infection, severe illness is more common in theelderly or immunocompromised individuals with chronic illnesses such asasthma, diabetes, kidney failure and heart disease. The annual epidemicsrun from November to March in the Northern Hemisphere and from April toSeptember in the Southern Hemisphere.

Avian influenza is caused by type A strains of influenza virus. Avianinfluenza occurs throughout the world. Infected birds may display a widerange of symptoms, from a mild illness to a highly contagious fataldisease. The highly contagious disease is caused by an especiallyvirulent strain of influenza virus. Infection by this strain isassociated with a sudden onset of severe symptoms, such as a lack ofenergy, decreased egg production, soft shelled eggs, a swelling of thehead, eyelids, etc., nasal discharge, coughing or diarrhea, resulting indeath (WHO, 2004). At present, 15 subtypes have been identified that caninfect birds but only H7, H5 and H9 subtypes are associated withoutbreaks. The current Asian and British Columbia outbreaks are causedby a H5N1 and H7N3 strains, respectively. As discussed above, influenzaviruses are a public health concern because these viruses lack amechanism for proofreading nucleic acid replication as well as a repairsystem for correcting such errors. Thus, influenza viruses areespecially prone to a high mutation rate during transcription.Additionally, influenza viruses are able to exchange or swap geneticmaterial from other subtypes from different species, thus allowingsubtypes to cross the species barrier that normally prevents the crossinfection of species specific viruses from one species to anotherunrelated species. This species barrier normally prevents avianinfluenza virus strains from infecting humans, but occasionally newstrains may have genetic material from both avian and human influenzavirus strains. This exchange of genetic material occurs when there is aclose proximity between humans and domestic poultry and swine. Swine mayact as a reservoir for both human and avian strains. Thus swine act as anatural incubator for the emergence of new strains that can infecthumans as well as avian species.

There are four antiviral drugs available in the US for the treatment ofinfluenza: amantadine, rimatadine, zanamivir (Zanamivir (Relenza™) andOseltamivir (Tamiflu™). Amantadine and rimatadine are effective onlyagainst influenza A. Amantadine, rimatadine and oseltamivir are approvedfor prophylaxis. Prophylaxis is indicated only for unvaccinated personsat high risk during an influenza outbreak. Antiviral agents have limiteduse due to poor tolerance and the occurrence of resistance. Presently,amantadine is the principal antiviral compound used against influenzainfection, but its activity is restricted to influenza A viruses. Theanti-neuraminidase inhibitors such as Zanamivir and Oseltamivir are anew class of antiviral agents licensed for use in the treatment of bothinfluenza A and B infections (Carr et al., Influenza Virus CarryingNeuraminidase with Reduced Sensitivity to Oseltamiver Carboxylate hasAltered Properties In Vitro and is Compromised for Infectivity andReplicative Ability In Vivo, Antiviral Res. 54: 79-88, 2002). Therefore,the development of new and effective antiviral drugs against influenza Aand B is of great clinical importance (Bamford, Neuraminidase Inhibitorsas Potential Anti-Influenza Drugs, J. of Enzyme Inhibition, Review 10:1-16, 1995).

Influenza vaccines are generally used before the onset of the influenzaseason and they are typically given to the segment of population that isconsidered to be at high risk. Vaccines come in several forms and theyaim at preventing or at least lessening the symptoms of disease.Vaccines are given prior to exposure of the virus to generateneutralizing antibodies against the strain that is most likely to causewide spread epidemics or pandemics. However, vaccinations can be costlyand stocks of the vaccine can be depleted quickly. Also, vaccines maynot contain the causative viral component. In other words, vaccineproduction depends upon estimating which strain will emerge as thedominant strain. Thus in any given year, there is only a limitedprotection against the various influenza strains. Furthermore, thetypical method of providing a vaccine via injection is unpleasant tomany. Prophylaxis treatments on the other hand, are used to preventinfection or lessen the severity of the disease post-exposure to thevirus. Oseltamivir™ as well as zanamivir or Relenza™ (Glaxo Wellcome,second generation antiviral) are neuraminidase inhibitors that block therelease of mature viral particles and thus prevent the infection ofneighboring cells. Neuraminidase inhibitors lesson the symptoms ofinfluenza infection and short the duration of the disease. Prophylaxismust be given within a 48-hour window of the onset of symptoms to beeffective and there is a risk of resistant strains emerging.

Severe acute respiratory syndrome (SARS) is the first major newinfectious disease of the 21st century. The first cases appeared inNovember of 2002 in Guangdong, China but it was only recognized as a newdisease in March of 2003. The spread of the disease was accelerated byinternational air travel such that cases were reported in 22 countries.However, with modern communication technologies and a globalcollaborative effort the disease was contained within four months ofbeing identified. The disease caused high morbidity and high mortalityrates, with symptoms including a high fever, headache, myalgia and a drycough. The mortality rate exceeded 60% in the over 60 age group (PeirisJ S et al., 2003). SARS was identified as being caused by a new virusthrough various laboratory techniques involving virus propagation intissue culture and electron microscopy studies. This was confirmed justdays later when the complete genome sequence was determined indicatingthat it was a new Coronavirus that was responsible. Therefore thedevelopment of antimicrobial drugs for use against this type ofinfectious disease is of great importance.

Other microorganisms that cause illness include the gram-positive andgram-negative bacteria such as Streptococcus, Staphylococcus, E. coli,Pseudomonas, and Haemophilus as well as fungal infections including theyeast, C. albicans. While active infection with these microorganisms isprimarily treated with antibiotics, some patients do not tolerateantibiotics well. Still others may wish to augment an antibiotictreatment with a treatment regiment that reduces or eliminates thesymptoms of bacterial or fungal infection such as sore throat. Stillothers may want to prevent or lessen the severity of infections by oneof these bacteria or fungi organisms by prophylactic treatment prior to,during or just after exposure.

Research interest has recently focused on various herbs, which containpotent antioxidant compounds that can provide significant protectionagainst chronic diseases and have antimicrobial or anti-tumour activity.Antioxidant substances such as flavonoids can be found in a variety ofherbs such as dandelion, ginger, green tea, and rosemary. It wasrecently reported that green-tea extract (GTE) inhibited the growth ofinfluenza A and B viruses in Madin-Darby canine kidney (MDCK) cells andin another study, Epigallocatechin-3-gallate (EGCG), one of thecomponents of green tea, inhibited the replication of HIV-1 (111B) andBal HIV strains in peripheral blood lymphocytes. These substances haveproven useful in the field treating various illnesses; however there hasnot been any progress in the creation of a prophylactic method for usewith antioxidant substances.

Therefore, there exists a need in the field to provide a prophylacticmethod for the reduction of the incidence of contracting an illnesscaused by a microbial organism.

SUMMARY OF THE INVENTION

Accordingly, it is an object of certain embodiments of the invention toprovide a method for reducing the incidence of contracting an illnesscaused by a microbial organism.

In the first aspect, the present invention relates to a method for theprophylactic use of an anti-microbial composition to reduce theincidence of contracting an illness. The method comprises the steps ofadministering to a mammal or a bird that has been, or will be, exposedto an illness caused by a microbe, an amount of an anti-microbialcomposition having a first ingredient obtainable from ginger; a secondingredient obtainable from green tea; and an acceptable carrier. Theamount of anti-microbial composition is effective, when administered, toreduce the incidence of contracting the illness.

In a second aspect of the invention, a prophylactic anti-microbialcomposition having a first ingredient obtainable from ginger, a secondingredient obtainable from green tea; and an acceptable carrier isdisclosed. The anti-microbial composition is effective, whenadministered as a nasal spray or as a throat spray to a mammal or a birdthat has been, or will be, exposed to an illness caused by a microbe, toreduce the incidence of contracting said illness.

These and various other advantages and features of novelty thatcharacterize the invention are pointed out with particularity in theclaims annexed hereto and forming a part hereof. However, for a betterunderstanding of the invention, its advantages, and the objects obtainedby its use, reference should be made to the accompanying descriptivematter, in which there is described a preferred embodiment of theinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a first aspect, the present invention relates to a composition. Thecomposition of the present invention includes ingredients that can beobtained from ginger, green tea and turmeric.

As used herein the term “flavors” includes both fruit and botanicalflavors.

As used herein the term “sweeteners” includes sugars, for example,glucose, sucrose and fructose. Sugars also include high fructose cornsyrup solids, invert sugar, sugar alcohols including sorbitol, andmixtures thereof. Artificial sweeteners are also included within thescope of the term, “sweetener.”

As used herein, the term “acceptable” means a component that is suitablefor use with humans and/or animals without undue adverse side effects(such as toxicity, irritation, and allergic responses), commensuratewith a reasonable risk/benefit ratio.

Further, as used herein, the term “safe and effective amount” refers tothe quantity of a component, which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic responses), commensurate with areasonable risk/benefit ratio when used in the manner described herein.

The term “inhibiting” a microbe, as used herein, refers to reducing orpreventing further growth of the microbe, or preventing the microbe fromattaching to normal cells, and/or the elimination of some or all of theinfectious particles from the human or animal being treated. Suitablemethods for determining microbe inhibition are discussed in theexamples.

The term “transmissivity” as used herein refers to the transfer of amicrobe from one host to another.

All active compounds used in the present invention may be obtained fromother sources, if available. Thus, the phrase “which can be obtainedfrom” or the phrase “which may be obtained from” is meant to encompasscompounds or compositions that are obtainable from turmeric, ginger, orgreen tea, and therefore encompasses synthetic forms of the samecompounds and/or compositions as well as the same compounds and/orcompositions obtained from other sources.

In a first embodiment, the composition of the present invention includesa first ingredient obtainable from ginger, and a second ingredientobtainable from green tea, in a safe and effective amount to provide oneor more of the beneficial effects described herein.

The first ingredient of the composition of the present invention may beobtained from ginger (Zingiber officinale, also commonly called gingerroot). Native to southern Asia, ginger is a 2- to 4-foot perennial thatproduces grass-like leaves up to a foot long and almost an inch wide.Ginger root, as it is called in the grocery store, actually consists ofthe underground stem of the plant, with its bark-like outer coveringscraped off.

The active compounds of ginger which may be employed in the presentinvention include, but are not limited to, 1,8-cineole,10-dehydrogingerdione, 10-gingerol, 6-gingerdione, 6-gingerol,6-shogaol, 8-β-17-epoxy-λ-trans-12-ene-15,16-diol, 8-gingerol,8-shogaol, 9-oxo-nerolidol, acetaldehyde, acetic acid, alanine,α-linolenic-acid, α-linolenic acid, α-phellandrene, α-piene,α-terpinene, α-terpineol, α-zingiberene, ar-curcumene, arginine,ascorbic acid, asparagine, β-bisabolol, β-carotene, β-elemene,β-eudesmol, β-ionone, β-myrcene, β-phellandrene, β-pinene, β-selinene,β-sesquiphellandrene, β-sitosterol, β-thujone, bornyl-acetate, boron,caffeic acid, calcium, camphene, camphor, capric acid, caprylic acid,capsaicin, caryophyllene, chavicol, chlorogenic acid, chromium, citral,citronellal, citronellal, cobalt, copper, cumene, curcumin, cystine,delphinidin, δ-cadinene, elemol, ethyl acetate, ethyl-myristate,farnesal, farnesene, ferulic acid, furfural, γ-aminobutyric acid,γ-terpinene, geranial, geraniol, geranyl-acetate, gingerenone, glutamicacid, glycine, hexahydrocurcumin, histidine, isogingerenone-B,isoleucine, kaempferol, lecithin, limonene, linoleic acid, magnesium,manganese, methionine, mufa, myrecene, myricetin, myristic acid, neral,nerol, nerolidol, niacin, nickel, oleic acid, oxalic acid, p-coumaricacid, p-cymene, p-hydroxy-benzoic acid, palmitic acid, pantothenic acid,paradol, patchoulic alcohol, phenylalanine, quercetin, riboflavin,selenium, shikimic-acid, terpinen-4-ol, thiamin, tryptophan, vanillicacid, vanillin, zinc, and zingerone. Also, mixtures of two or more ofthese active compounds may be employed.

The first ingredient of the composition of the present invention, whichmay be obtained from ginger, can be incorporated in the composition ofthe present invention in many different forms including extracts such asginger powder extracts, ginger fluid extracts, ginger powder includingginger root powder, and one or more active compounds of ginger, partsof, or whole ginger plants, tinctures thereof, and mixtures thereof.Preferably, the first ingredient of the composition of the presentinvention is selected from ginger extract, and ginger root powder.

Each gram of the composition of the present invention preferablycontains about 1 mg to about 150 mg of ginger root powder. Mostpreferably, each gram of the composition contains about 6 mg to about110 mg of ginger root powder. These ranges use, as a baseline, the useof Ginger Root Powder, ex. Stryka Botanics in the ingested formulationand Ginger Extract K (Aquaresin® Ginger), ex. Kalsec®, Inc. ofKalamazoo, Mich. in the spray formulation.

The amounts of various ingredients are given herein in terms of one formof the ingredient, i.e. ginger root powder. If that ingredient ispresent in another form, then the amount to be employed is that amountwhich will provide the same amount of the one or more active compoundsas the amount of that ingredient given herein. For example, if atincture of ginger is employed, the amount of the tincture employed willbe the amount that provides the same amounts of one or more activecompounds as would be provided by the amounts of ginger root powderspecified above. This applies to all ingredients for which the amountsare given herein for one particular form of that ingredient.

The second ingredient of the composition of the present invention may beobtained from green tea. The second ingredient obtained from green teamay have an antioxidant effect. Green tea is the dried leaves and leafbuds of the shrub Camellia sinensis. It is mainly produced in China andJapan. Dried tea leaves are composed mainly of phytochemicals known aspolyphenols (about 36%), principally flavonols (including catechins),flavonoids, and flavondiols. The leaves also contain plant alkaloids(about 4%), including caffeine, theobromine and theophylline.

The pharmacological activities of green tea are mainly due to its activecompounds. The active compounds of green tea useful in the presentinvention include, but are not limited to, flavonols, catechins,flavonoids, flavondiols, plant alkaloids, caffeine, theobromine,theophylline, phenolic acids, proteins, carbohydrates, and minerals.

The second ingredient which may be obtained from green tea, can beincluded in the composition in the form of green tea powder, green teaextracts such as green tea powder extracts, green tea fluid extracts,and one or more active compounds of green tea, part of, or whole greentea plants, green tea leaves, tinctures thereof, or mixtures thereof.Preferably, the second ingredient of the composition of the presentinvention is selected from green tea leaves, green tea powder and greentea extract. More preferably, the second ingredient of the compositionof the present invention is green tea extract.

Each gram of the composition of the present invention preferablycontains about 1 mg to about 20 mg of green tea extract. Mostpreferably, each gram of the composition contains about 4 mg to about 15mg of green tea extract. These ranges use, as a baseline, the use ofGreen Tea, ex. Stryker Botanics in the ingested formulation and GreenTea Extract, ex. Phytoway, Inc., ChanSha, P.R. China, in the sprayformulation.

The ingredients of the composition of the present invention, which maybe obtained from ginger and green tea, and turmeric, may be used in theforms of turmeric powder, ginger powder and green tea powder, each ofwhich may be ground from the rhizome of turmeric, ginger root and greentea leaves, respectively. For a particular active compound of ginger,green tea or turmeric, for which a synthetic route is known, the activecompound may be synthesized. The plant extracts, if desired, may beprepared as described below. Alternatively, turmeric powder, gingerpowder, green tea powder and/or one or more of the active compoundscontained therein may be purchased from commercial sources such as theKelsec®, Inc. of Kalamazoo, Mich.

The plant extracts, e.g., turmeric extract, ginger extract, green teaextract and horseradish extract that may be used in the compositions ofthe invention, may be produced using common extraction procedures.Alternatively, the extracts may be purchased from commercial sourcessuch as the Kelsec®, Inc. of Kalamazoo, Mich.

The processes for the preparation of pharmacologically or biologicallyactive plant extracts in a convenient, administrable dosage form fromany of the plants mentioned above, are well known in the art.

The composition of the present invention may be used to treat viralinfection, since the composition of the present invention hassignificant antimicrobial properties as demonstrated by the examples ofthis application. The composition of the present invention may also beused as a therapeutic composition to treat one or more symptoms of aviral infection, including sore throat, congestion, laryngitis,mucositis, and/or mucous membrane inflammation by administration to apatient suffering from one or more of these symptoms or ailments.

The composition of the present invention may also be employed to reducethe incidence of contracting an illness. In this application of thecomposition of the present invention, a safe and effective amount of thecomposition of the present invention is administered to a mammal or abird that has been or will be exposed to an illness caused by a microbe,to reduce the incidence of contracting said illness, relative to amammal or a bird that has been or will be exposed to an illness causedby a microbe to which the composition of the present invention has notbeen administered.

Preferably, the composition of the present invention may be formulatedin any acceptable dosage form including, but not limited to, capsules,tablets, lozenges, troches, hard candies, powders, sprays, gels,elixirs, syrups, and suspensions or solutions. The composition of thepresent invention may also be administered in the form of a nutritionalsupplement, in which case the composition of the invention may be thenutritional supplement or may form a part of a nutritional supplementcontaining additional ingredients.

The composition of the present invention may also be formulated with anacceptable carrier. The acceptable carrier may include, but is notlimited to: (a) carbohydrates including sweeteners, more preferably,fructose, sucrose, sugar, dextrose, starch, lactose, maltose,maltodextrins, corn syrup solids, honey solids, commercial tabletnutritional supplements including Emdex™, Mor-Rex™, Royal-T™, Di-Pac™,Sugar-Tab™, Sweet-Rex™, and New-Tab™; (b) sugar alcohols includingmannitol, sorbitol and xylitol; and (c) various relatively insolubleexcipients including dicalcium phosphate, calcium sulfate, calciumcarbonate, microcrystalline cellulose and other tableting ingredients.

Lozenges, tablets, and troches in this invention may differ in shape,size and manufacturing technique. In the case of tablets, for oral use,the acceptable carrier may further include lactose and corn starch.Lubricating agents may also be added to the tablets, including, forexample, magnesium stearate, sodium lauryl sulfate and talc. Tablets mayalso contain excipients such as sodium citrate, calcium carbonate andcalcium phosphate. Disintegrants such as starch, alginic acid andcomplex silicates, may also be employed. Tablets may also includebinding agents such as polyvinylpyrrolidone, gelatin, PEG-8000 and gumacacia.

In the case of lozenges for oral use, the common acceptable carrier mayfurther include a binder such as PEG-8000. Preferably lozenges weighabout 0.1 to about 15 grams to provide a suitable dissolution rate whentaken orally. More preferably, lozenges weigh about 1 to about 6 grams.

The production of lozenges is well known in the art and anyone withordinary skill in the art can readily produce lozenges with thecompositions of the present invention. The composition is preferablystored in an airtight container and in a cool dark place.

Tablets and troches can be manufactured using procedures known in theart with minor changes in the optional ingredients. Such changes arewithin the skill of the ordinary skilled artisan.

Alternatively, the composition of the present invention may beformulated in liquid form, such as syrups, mouthwashes or sprays, with asolvent or dispersant such as water, or other liquids and optionally ina pharmaceutically acceptable carrier, for repeated delivery of thecomposition to oral and oropharyngeal mucous membranes over a sustainedperiod of time. Preferably, the treatment time is about 5 to 60 minutes,and more preferably about 20 to 30 minutes, so as to permit a prolongedcontact of the composition with mouth and throat tissues. Alternatively,such formulations can be in a concentrated form suitable for dilutionwith water or other materials prior to use.

The composition may also be formulated in chewable forms, such as softcandy, gum drops, liquid filled candies, and chewing gum bases, or inthe form of dental products, such as toothpastes and mouthwashes. Inuse, the chewable composition is preferably retained in the mouth over asustained period of time of preferably about 5 to 60 minutes, and morepreferably about 20 to 30 minutes. Dental products may be used in theordinary manner of using such products.

The composition of the invention may be formulated in capsule form, withor without diluents. For capsules, useful diluents include lactose anddried corn starch. When suspensions are employed, emulsifying and/orsuspending agents may be employed in the suspensions. In addition, solidcompositions including one or more of the ingredients of the lozengesdescribed above may be employed in soft and hard gelatin capsules.

The composition of the present invention may also be formulated into anasal aerosol or inhalant composition. Such a composition may beprepared using well-known techniques. For these types of formulations,suitable carriers may include the following ingredients: saline with oneor more preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or conventional solubilizing or dispersion agents.

Other materials, which may optionally be included in the composition ofthe present invention, include resveratrol (trihydroxystilbene),inositol, other B-complex vitamins, and additional anti-inflammatories.Also, ingredients such as sweeteners, flavorants, coloring agents, dyes,preservatives, emulsifying agents, suspending agents, melting agents,excipients, demulcents and solvents or diluents such as water, ethanol,propylene glycol, glycerin and various combinations thereof, may beincluded in the composition of the present invention.

In an optional embodiment, the composition of the present inventionincludes one or more ingredients obtainable from turmeric, in a safe andeffective amount to provide one or more of the beneficial effectsdescribed herein. Turmeric (Curcuma longa), or Haldi in Hindi, is usedvery widely as medicine as well as a common ingredient in Indiancooking. The rhizome of turmeric is used in medicine and food as a finepowder.

The yellow pigment of the rhizome of turmeric is composed of threecompounds known as curcuminoids. The three curcuminoids are curcumin(diferuloylmethane), desmethoxycurcumin (hydroxycinnamoylferuloylmethane), and bis-desmethoxycurcumin (dihydroxydicinnamoylmethane) (see Drug Analysis, Chromatography and Microscopy, p. 169, AnnArbor Science Inc., 1973). The essential oils of turmeric (Curcumalonga) are primarily composed of the following compounds: d-camphor(about 1%), cyclo-isoprenemyrcene (about 85%), and p-tolylmethylcarbinol(about 5%), (see E. Gunther, The Essential Oil, pp. 123-4, Van NostrandCo., 1955).

The ingredient of the composition of the present invention, obtainedfrom turmeric, preferably includes curcuminoids, such as curcumin(diferuloylmethane), desmethoxycurcumin (hydroxycinnamoylferuloylmethane), and bis-desmethoxycurcumin (dihydroxydicinnamoylmethane), and mixtures of two or more of these curcuminoids.

Methods for isolating curcuminoids from turmeric are known (see Janakiand Bose, An Improved Method for the Isolation of Curcumin FromTurmeric, J. Indian Chem. Soc. 44: 985, 1967). Alternatively,curcuminoids for use in the present invention can be prepared bysynthetic methods.

The ingredient, which can be obtained from of turmeric, can beincorporated into the composition of the present invention in a varietyof different forms. Those different forms preferably include extracts ofturmeric such as turmeric powder extracts, turmeric fluid extracts, oneor more the curcuminoid compounds, and turmeric powder, parts of, orwhole plants of turmeric, tinctures thereof, and mixtures thereof. Morepreferably, the optional ingredient obtainable from turmeric is aturmeric extract.

When the ingredient obtainable from turmeric is used, each gram of thecomposition of the present invention preferably contains about 1 mg toabout 20 mg of turmeric powder extract. Most preferably, each gram ofthe compositions contains about 6 mg to about 15 mg of turmeric powderextract. These ranges are based on the use of Turmeric Extract 95%, ex.Pharmline, Inc. in the ingested formulation and Turmeric Root Extract(Oleoresin Turmeric), ex. Kalsec®, Inc., Kalamazoo, Mich., in the sprayformulation.

Also, the composition of the present invention may include one or moreingredients obtainable from horseradish root, in a safe and effectiveamount to provide one or more of the beneficial effects describedherein.

The optional ingredient obtainable from horseradish root may includeextracts from the Cochlearia Armoracia. Horseradish contains volatileoils that are similar to those found in mustard. These includeglucosinolates (mustard oil glycosides), gluconasturtiin, and sinigrin,which yield allyl isothiocynate when broken down in the stomach.

Ethanol, propylene glycol and glycerin and various combinations thereof,may be optionally included in the composition of the present invention,up to about 10 percent by weight of the total as additional activeingredients. Most preferably, up to about 10 percent per total weightethanol is added as an active ingredient. Even more preferable, 2.5 to 7percent ethanol is added.

The optional sweeteners which may be used in the composition of thepresent invention include, but are not limited to, saccharin, aspartame,cyclamates, acesulfame K, neohesperidin dihydrochalcone, other supersweeteners, and mixtures thereof, which may be added to the carrier inamounts sufficiently low so as not to chemically interact with the mainingredients of the composition.

The optional flavorants which may be used in the composition of thepresent invention include, but are not limited to, peppermint,peppermint-menthol, eucalyptol, wintergreen, licorice, clove, cinnamon,spearmint, cherry, lemon, orange, lime, menthol and various combinationsthereof.

Preferably, the main ingredients described above, that may be derivedfrom ginger, green tea and, optionally, turmeric, make up from about 0.5to about 90% by weight of the total composition. More preferably, themain ingredients will make up about 10 to about 70% by weight of thetotal composition. Most preferably, the main ingredients make up about20 to about 40% by weight of the total composition.

The non-carrier ingredients of the composition, including theingredients obtainable from turmeric, ginger, and green tea as discussedabove, can be increased or decreased proportionally in the compositionof the present invention depending on the amount of carrier used in thecomposition, without substantially affecting the effectiveness of thecomposition for its intended use.

Reducing or preventing transmission relates to preventing or reducingthe spread of a microbe from one patient (infected) to another patient(non-infected). Some patients may be considered carriers of theinfection. Carriers are individuals who actively shed microbes but donot suffer from an acute infection. These carriers may be said to bepersistently (or chronically) infected with the microbe. In addition tothe persistently infected shedder, other infective individuals may bethose who are actively infected, and particularly those in the early orlate stages of an acute infection. One aspect of the invention relatesto administering to a mammal or a bird infected with a microbe, acomposition of the present invention, to prevent the spread of thedisease to other mammals or birds and/or reduce the symptoms of thedisease in the infected mammal or bird.

Prophylactic treatment is aimed at a patient that will soon be exposedto a microbe or has recently been exposed to a microbe. Suchprophylactic treatment may be effective either alone, or to augment avaccine. Prophylactic treatment may also be used against microbes forwhich there is not yet a vaccine available. In the case of prophylactictreatment, the composition of the invention is administered to a patientthat will be exposed to a microbe or has recently been exposed to amicrobe for the purpose of reducing the incidence of active infection bythe microbe in that patient.

In another aspect, the present invention relates to a method ofreducing, treating or preventing of at least one symptom or adverseeffect of viral infection by administering, to a patient infected with avirus, a composition of the present invention, including ingredientsthat can be obtained from ginger and green tea.

In the method, the patient may be a human, an in vitro cell system, oran animal. Preferably, the patient is a mammal, more preferably, ahuman. In the method, the virus that may be inhibited by administrationof the composition of the present invention includes, among otherviruses, rhinoviruses, influenza viruses, West Nile virus, herpessimplex virus, HIV-1, HIV-2, adenovirus, cornavirus, influenza virus,rubella virus, yellow fever virus and respiratory syncytial virus (RSV).In a preferred embodiment, the viruses that may be inhibited byadministration of the composition include at least human rhinovirus 16,Herpes I Virus (HSV-1), Influenza A/Moscow/10/99, avian influenza A(H5N1), and B/Guangdong/120/00.

Alternatively, the patient may be a member of the bird (Avian) species,which includes the common commercial poultry birds: turkeys, ducks,geese and chickens, less commonly the ostrich as well as other birdspecies that are commonly kept as house pets, for example canaries andparrots. The composition may be administered by directly spraying thecomposition into the nasal passage of the bird or the composition may beadministered by creating a mist through which the birds walk. Thus, thecomposition may be given prophylactically to act in a virucidal orvirustatic manner. Alternatively, the composition may be used to reducethe transmissivity of the virus.

The symptoms, caused by a viral infection, that may be treated, reduced,or at least partially prevented by this method of the present invention,may include one or more of headache, joint pain, fever, cough, sneezing,muscle ache, running nose, dry mouth, dizziness, and other symptomsrelated to viral infection. In birds, these symptoms include a lack ofenergy, decreased egg production, soft shelled eggs, a swelling of thehead, eyelids, etc., nasal discharge, coughing or diarrhea.

In the method, microorganisms that may be inhibited by administration ofthe composition of the present invention include gram-positive bacteriasuch as Streptococcus, Staphylococcus, gram-negative bacteria such as E.coli, Pseudomonas, Haemophilus and fungi such as Histoplasma andBlastomycosis and yeast such as C. albicans and Crytococcus.

The effective amount of the composition will vary depending on suchfactors as the patient being treated, the particular mode ofadministration, the activity of the particular active ingredientsemployed, the age, bodyweight, general health, sex and diet of thepatient, time of administration, rate of excretion, the particularcombination of ingredients employed, the total content of the mainingredient of the composition, and the severity of the illness orsymptom. It is within the skill of the person of ordinary skill in theart to account for these factors.

The composition may be administered about 1 to about 15 times per day,as needed, more preferably, about 2 to about 12 times per day, asneeded, or most preferably, about 6 to about 10 times per day, asneeded. The composition of the present invention may be administered inany acceptable dosage form, as described above, including, but notlimited to, tablets, capsules, lozenges, troches, hard candies, powders,oral sprays, nasal sprays, gels, elixirs, syrups, chewable compositions,dental products, suspensions, and solutions.

Each dosage of the composition contains a safe and effective amount ofthe composition of the present invention. An effective amount for eachtherapeutic administration contains a total of about 0.1 gram to about 1gram of the ingredients, which may be obtained from ginger and greentea. More preferably, an effective amount of the composition for eachtherapeutic administration contains a total of about 0.2 gram to about0.5 gram of the ingredients which may be obtained from ginger and greentea. The amounts of the various ingredients of the compositionadministered in accordance with the method of the present invention arethe same as given above for the composition of the present invention.

Preferably, during each oral administration of the composition, thecomposition is held in the mouth for at least about 5 to about 60minutes to enable the main ingredients of the composition to contact themouth tissue or throat before it completely dissolves. More preferably,the composition is held in the mouth for at least about 15 to about 30minutes.

When the composition is administered as a spray, the amounts each of theactive ingredients may be reduced as the spray composition delivers theactive ingredients more directly to the location where they are needed,as compared to a lozenge or capsule for example.

The following preferred ranges define compositions according to theinvention that are suited for administration in a spray formulationaccording to the methods of the invention.

Each gram of the composition administered in a spray according to themethods of the present invention preferably contains about 1 mg to about10 mg of aquaresin® ginger. Most preferably, each gram of thecomposition contains about 3 mg to about 7 mg of aquaresin® ginger.

Each gram of the composition administered in a spray according to themethods of the present invention preferably contains about 1 mg to about20 mg of green tea leaf extract. Most preferably, each gram of thecomposition contains about 4 mg to about 15 mg of green tea leafextract.

Each gram of an optional embodiment of a composition administered in aspray according to the methods of present invention preferably containsabout 1 mg to about 12 mg of soluble oleoresin turmeric. Mostpreferably, each gram of the composition contains about 4 mg to about 9mg of soluble oleoresin turmeric.

The invention will be further illustrated by the examples given belowwhich are not to be construed as limiting the invention in any way. Thescope of the invention is to be determined by the claims appendedhereto.

EXAMPLE 1 A Composition of the Present Invention

A composition of the present invention formulated in the form oflozenges was prepared using the procedure described above. Theingredients of the lozenge are listed below: Sugar  1 g  Slippery elmbark 118 mg  Turmeric extract (5% curcumin) 18 mg Ginger root 140 mg Horseradish root 70 mg Green tea leaf extract (30% catechin andpolyphenols) 14 mg

EXAMPLE 2 Treatment of Sore Throat

Each of seven patients, suffering from sore throats, ingested onelozenge formulated according to Example 1 every two hours by holding thelozenge in his or her mouth for about 15-30 minutes until the lozengecompletely dissolved. No patient took more than 10 lozenges in any givenday.

The patients that were treated reported complete relief from thesymptoms of their sore throats after ingesting from 2 to 20 lozenges. Itwas also found that each lozenge can provide relief from a sore throatfor up to 6 hours.

EXAMPLE 3 In Vitro Testing of Virucidal Activity of the Composition

The in vitro testing protocol for virucidal activity employed in thisexample uses human rhinovirus 16 (hereafter “HRV-16”) as the targetvirus, and the MRC-5 cell line related to human tissues described byJacobs et al., Characteristics of Human diploid MRC-5, Nature (London),227: 168-170 (1970) as the host cell for the HRV-16 viruses. Residualvirus infectivity following incubation of the test substances with thevirus was titrated on the MRC-5 cell line for rhinovirus growth byvisually scoring the cytopathic effect (CPE) induced by virusreplication through microscopic observation. More specifically, CPE wasscored by observing ballooning/rounding cells in the MRC-5 culture.

To determine the virucidal activity, the composition of Example 1(hereafter “Substance 1”), was employed at an initial dilution of 1/20and then further diluted by serial dilutions in saline. The dilutedcompositions were incubated with HRV-16 for a set time period and thenthe reaction was terminated by adjustment to a neutral pH with cellinfection media. The resultant solution was then titrated out on MRC-5cells at a dilution of 1/10 across a testing plate to carry out theinfection of the cells. Each plate housed a virus control, whichcontained only HRV-16 infected MRC-5 cells, and a cell control, whichcontained only uninfected MRC-cells.

The plates were further incubated for 4 days after the infection.Residual viral infectivity was measured using the assay discussed above.From the results shown in Tables 1-4, all of the controls on the plateworked well.

From the assay, it was concluded that Substance 1, at a 1/20 dilution,was effective in producing an HRV-16 viral log reduction of 1.50 (−log₁₀TCID₅₀) at the 1-minute incubation period. A 1/40 dilution of Substance1 produced a log reduction of 1.00 (−log₁₀ TCID₅₀) also at the 1-minuteincubation period. After the 2-minute and 5-minute incubation periods, ½log reductions in HRV-16 titer were achieved. Therefore, these resultstend to indicate that a 1-minute contact time between Substance 1 andHRV-16 would produce the most effective viral titer reduction.

Table 1 shows the residual virus titers and log reductions of infectiousRhinovirus 16 on MRC-5 cells at one termination time point, of Substance1 at different dilutions. TABLE 1 pH value of pH value of 1 MinuteIncubation Substance 1 in terminated Virus Control Residual Virus LogReductions Dilutions Isotonic solution solution (TCID₅₀) titer (TCID₅₀)(TCID₅₀) 1/20 5.03 7.73 3.80 2.30 1.50 1/40 5.13 7.77 3.80 3.30 0.501/80 4.98 7.83 3.80 3.80 0.00 1/160 4.98 7.73 3.80 3.80 0.00

Tables 2-4 show the results of a second trial on the residual virustiters and the log reductions of infectious HRV-16 on MRC-5 cells atthree different termination time points, of Substance 1 at differentdilutions. TABLE 2 1 Minute Incubation HRV-16 Residual HRV-16 Dilutionsof Control Titer HRV-16 titer log Reductions Substance 1 (TCID₅₀)(TCID₅₀) (TCID₅₀) 1/20 3.30 1.80 1.50 1/40 3.30 2.30 1.00 1/80 3.30 2.800.50 1/160 3.30 2.80 0.50 1/320 3.30 2.80 0.50

TABLE 3 2 Minute Incubation HRV-16 Residual HRV-16 Dilutions of ControlTiter HRV-16 titer log Reductions Substance 1 (TCID₅₀) (TCID₅₀) (TCID₅₀)1/20 3.30 2.80 0.50 1/40 3.30 2.80 0.50 1/80 3.30 2.80 0.50 1/160 3.302.80 0.50 1/320 3.30 2.80 0.50

TABLE 4 5 Minute Incubation HRV-16 Residual HRV-16 Dilutions of ControlTiter HRV-16 titer log Reductions Substance 1 (TCID₅₀) (TCID₅₀) (TCID₅₀)1/20 3.30 2.80 0.50 1/40 3.30 2.80 0.50 1/80 3.30 3.30 0.00 1/160 3.302.80 0.50 1/320 3.30 2.80 0.50In Tables 1-4, TCID50 = −log₁₀ TCID₅₀.

Similar virucidal tests have been carried out for Substance 1 usingother viruses, including Herpes I Virus (HSV-1) using Vero cells as thehost cell, Influenza A/Moscow/10/99, and B/Guangdong/120/00 using MDCKcells as the host cell. The results on these virucidal tests aresummarized below in Tables 5-13.

Tables 5-7 show the residual virus titers and log reductions ofinfectious HSV-1 on Vero cells at three different termination timepoints, of Substance 1 at different dilutions. TABLE 5 1 MinuteIncubation HSV-1 Residual HSV-1 Dilutions of Control Titer HSV-1 titerlog reductions Substance 1 (−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) 1/40 3.80 0.00 3.80 1/80 3.80 0.00 3.80 1/160 3.80 2.80 1.001/320 3.80 2.80 1.00 1/640 3.80 2.80 1.00

TABLE 6 2 Minute Incubation HSV-1 Residual HSV-1 Dilutions of ControlTiter HSV-1 titer log reductions Substance 1 (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) 1/40 3.80 0.00 3.80 1/80 3.80 0.00 3.80 1/1603.80 1.80 2.00 1/320 3.80 2.80 1.00 1/640 3.80 2.80 1.00

TABLE 7 5 Minute Incubation HSV-1 Residual HSV-1 Dilutions of ControlTiter HSV-1 titer log reductions Substance 1 (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) 1/40 3.80 0.00 3.80 1/80 3.80 0.00 3.80 1/1603.80 1.80 2.00 1/320 3.80 2.80 1.00 1/640 3.80 2.80 1.00

Tables 8-10 show the residual virus titers and log reductions ofinfluenza A/Moscow/10/99 at three different termination time points, ofSubstance 1 at different dilutions. TABLE 8 1 Minute Incubation A/MoscowResidual A/Moscow Dilutions of Virus Titer A/Moscow titer log reductionsSubstance 1 (−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) 1/10 2.800.00 2.80 1/20 2.80 0.00 2.80 1/40 2.80 1.80 1.00 1/80 2.80 1.80 1.001/160 2.80 1.80 1.00 1/320 2.80 1.80 1.00 1/640 2.80 1.80 1.00 CitrateBuffer 2.80 1.80 1.00

TABLE 9 2 Minute Incubation A/Moscow Residual A/Moscow Dilutions ofVirus Titer A/Moscow titer log reductions Substance 1 (−log₁₀ TCID₅₀)(−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) 1/10 2.80 0.00 2.80 1/20 2.80 0.00 2.801/40 2.80 1.80 1.00 1/80 2.80 1.80 1.00 1/160 2.80 1.80 1.00 1/320 2.801.80 1.00 1/640 2.80 1.80 1.00 Citrate Buffer 2.80 1.80 1.00

TABLE 10 5 Minute Incubation A/Moscow Residual A/Moscow Dilutions ofVirus Titer A/Moscow titer log reductions Substance 1 (−log₁₀ TCID₅₀)(−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) 1/10 2.80 0.00 2.80 1/20 2.80 0.00 2.801/40 2.80 1.80 1.00 1/80 2.80 1.80 1.00 1/160 2.80 1.80 1.00 1/320 2.801.80 1.00 1/640 2.80 1.80 1.00 Citrate Buffer 2.80 0.00 2.80

Tables 11-13 show the residual virus titers and log reductions ofInfluenza B/Guangdong/120/00 at three different termination time points,of Substance 1 at different dilutions. TABLE 11 1 Minute IncubationB/Guangdong Residual B/ B/Guangdong log Dilutions of Virus TiterGuangdong titer reductions Substance 1 (−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀)(−log₁₀ TCID₅₀) 1/10 1.80 0.00 1.80 1/20 1.80 0.00 1.80 1/40 1.80 1.800.00 1/80 1.80 1.80 0.00 1/160 2.30 1.80 0.50 1/320 2.30 1.80 0.50 1/6401.80 2.30 −0.50 Citrate 1.80 0.00 1.80 Buffer

TABLE 12 2 Minute Incubation B/Guangdong Residual B/ B/Guangdong logDilutions of Virus Titer Guangdong titer reductions Substance 1 (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) 1/10 1.80 0.00 1.80 1/20 1.800.00 1.80 1/40 1.80 1.80 0.00 1/80 1.80 1.80 0.00 1/160 2.30 1.80 0.501/320 2.30 1.80 0.50 1/640 1.80 2.80 −1.00 Citrate 1.80 0.00 1.80 Buffer

TABLE 13 5 Minute Incubation B/Guangdong Residual B/ B/Guangdong logDilutions of Virus Titer Guangdong titer reductions Substance 1 (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) 1/10 1.80 0.00 1.80 1/20 1.800.00 1.80 1/40 1.80 1.80 0.00 1/80 1.80 1.80 0.00 1/160 2.30 1.80 0.501/320 2.30 1.80 0.50 1/640 1.80 2.80 −1.00 Citrate 1.80 0.00 1.80 Buffer

As can be seen from above results, Substance 1 is effective ininhibiting or exterminating influenza viruses and human rhinoviruses. Asa result, Substance 1 should be effective in treating influenza andcommon colds.

EXAMPLE 4 In Vitro Testing of Virustatic Activity of the Composition

The in vitro testing protocol for virucidal activity employed in thisexample used human rhinovirus 16 (HRV-16) as the target virus, and therhinovirus sensitive Hela cell line related to human tissues describedby Conant et al., (Basis for a Numbering system. I. Hela cells forPropagation and Serologic Procedure, J. Immunol., 100: 107-113, 1968) asthe host cell for the HRV-16 virus.

Substance 1 was dissolved in infection media to the following dilutions:1/20, 1/40, 1/80, 1/160 and 1/320. These dilutions were incubated onplates of MRC-5 cells for 30 minutes at 37° C. (5% CO₂). After theincubation period, each Substance 1 dilution with MRC-5 cells in a wellof the plates was subjected to HRV-16 at a known titer of 2.30 (−log₁₀TCID₅₀). Each plate housed a virus control (the Hela cells infected withHRV-16 viruses and without Substance 1), a cell control (Hela cellsonly) and the test compound controls at the different dilutions (Helacells with the test substance only). All the other samples on the platecontained the Hela cells infected with HRV-16 viruses and Substance 1 atdifferent dilutions. The plates were further incubated for 4 days afterinfection.

Residual virus infectivity following incubation of Substance 1 with thevirus was titrated on the Hela cell line for rhinovirus growth bymeasuring the cytopathic effect (CPE) induced by the virus using thefollowing procedure.

The remaining viable Hela cells after incubation with Substance 1 werestained with crystal violet solution. Excess crystal violet was removedby washing and the crystal violet stained cells were solubilized using amixture of methanol and acetic acid. The absorbance of the solution wasthen measured at 540 nm in an ELISA plate reader. The level of virusinduced CPE was inversely proportional to the absorbance.

The results generated from the crystal violet assay enabled the toxicconcentration and the effective concentration of Substance 1 to bedetermined by fitting an equation, y=m×+c, wherein x corresponds to thedilution of Substance 1 and y corresponds to percentage of toxicity ofSubstance 1 to the cells. From this equation, the TC₅₀ (concentration atwhich Substance 1 indicates 50% toxicity to the cells) is at a 1/571dilution of Substance 1.

This result correlates well with the percentage of cell survivors atvarious dilution of Substance 1, which was also measured using thecrystal violet assay, as shown in Table 14 below. TABLE 14 Dilution ofSubstance 1 without Virus % Cell Survivors 1/320 89.7 1/160 94.6 1/8097.6 1/40 109.3 1/20 168.2

Using the same equation, wherein x still corresponds to the dilution ofSubstance 1 and y corresponds to the percent efficacy of Substance 1 inthe presence of the virus, the EC₅₀ (concentration at which the testsubstance indicates 50% efficacy in the presence of virus) wasdetermined to be at a 1/91 dilution of Substance 1. This resultcorrelates well with the percentage of viable cells at various dilutionsof Substance 1 measured using the crystal violet assay, as shown inTable 15 below. TABLE 15 Substance 1 dilution and Virus % Viable Cells1/320 + HRV-16  79.3 1/160 + HRV-16  62.3 1/80 + HRV-16 39.0 1/40 +HRV-16 15.9 1/20 + HRV-16 −220.0

In Tables 14 and 15, % Cell Survivors=(Compound only/Cell only)×100; and% Viable Cells=(Cell only−Compound+Virus)/(Cell only−Virus only)×100.

“Compound only” denotes the measurement results for the wells containingonly Hela cells and Substance 1 at a predetermined dilution.

“Cell only” denotes the measurement results for the wells containingonly uninfected Hela cells.

“Compound+Virus” denotes the measurement results for the wellscontaining the Hela cells infected with HRV-16 viruses and Substance 1at a predetermined dilution.

“Virus Only” denotes the measurement results for the wells containingthe Hela cells infected with HRV-16 only.

EXAMPLE 5 An Antimicrobial Lozenge of the Present Invention

An antimicrobial lozenge was made according to the formulation set forthbelow. 1) Dextrose 865.0 mg 2) Slippery Elm Bark 150.0 mg 3) StearicAcid 75.0 mg 4) Ginger Root 105.0 mg (Children) or 140.0 mg (Adult) 5)Horseradish Root 70.0 mg 6) Honey Natural Flavor 40.0 mg 7) TurmericExtract (5% Curcumin) 15.0 mg 8) Green Tea Leaf Extract (36% C & P) 14.0mg 9) Silicon Dioxide 14.0 mg 10) Magnesium Stearate 12.0 mg 11)Sucralose/Splenda 4.0 mg Tablet Weight: 1364.0 mgNote: C & P as used herein means “catechols and phenols.”

EXAMPLE 6 An Antimicrobial Spray of the Present Invention

An antimicrobial spray was made according to the formulation set forthbelow. (1) Slippery Elm Bark Extract 18.52 mg (2) Oleoresin Turmeric,Soluble (˜8.5% Curcumin) 8.82 mg (3) Aquaresin ® Ginger 7.0 mg (4)Horseradish Flavor WONF 0.62 mg (5) Green Tea Leaf PE 50% Colorimetric14.0 mg (6) Honey Natural Flavor 40.0 mg (7) Ethanol (95%) @ 5% 68.2 mg(8) Glycerine 603.42 mg (9) Distilled Water 603.42 mg Total Weight:1364.0 mg

EXAMPLE 7 In Vitro Testing of Antimicrobial Lozenge

The antimicrobial lozenge of Example 5 was tested for virucidal andvirustatic activity against infection of MDCK cells with influenzaviruses of the strains A/NewCaledonia/20/99 (H1N1), A/Panama/2007/99(H3N2), and B/Guangdong/120/00.

In determining virucidal activity, the lozenge was tested at dilutionsof 1/10, 1/20, 1/40, 1/80, 1/160, 1/320, and 1/640. The lozenge wasdiluted with saline isotonic solution (Normasol). Each dilution wastested at termination points of 1, 2, and 5 minutes after the lozengecame in contact with each virus. The reaction was terminated with 1.8 mlof 0% FBS cell media.

The log reductions in this example are reported as −log₁₀ TCID₅₀ andwere calculated using the Karber equation. TABLE 16 The residual virustiters and log reductions of infectious A/New Caledonia/20/99 (H1N1)virus after the 1-minute termination time point at different dilutions.1 Minute Incubation A/New Caledonia Residual Influenza Virus log Virustiter titer reductions Dilution (−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) 1/10 2.80 0.00 2.80 1/20 2.80 2.30 0.50 1/40 2.80 1.80 1.00 1/802.80 2.30 0.50 1/160 2.80 1.80 1.00 1/320 2.80 1.80 1.00 1/640 2.80 1.801.00 Citrate 2.80 1.80 1.00 Buffer

TABLE 17 The residual virus titers and log reductions of infectiousA/New Caledonia/20/99 (H1N1) virus after the 2-minute termination timepoint at different dilutions. 2 Minute Incubation A/New CaledoniaResidual Influenza Virus log Virus Titer titer reductions Dilution(−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) 1/10 2.80 0.00 2.80 1/202.80 1.80 1.00 1/40 2.80 1.80 1.00 1/80 2.80 1.80 1.00 1/160 2.80 1.801.00 1/320 2.80 1.80 1.00 1/640 2.80 1.80 1.00 Citrate 2.80 1.80 1.00Buffer

TABLE 18 The residual virus titers and log reductions of infectiousA/New Caledonia/20/99 (H1N1) virus after the 5-minute termination timepoint at different dilutions. 5 Minute Incubation A/New CaledoniaResidual Influenza Virus log Virus titer titer reductions Dilution(−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) (−log₁₀ TCID₅₀) 1/10 2.80 0.00 2.80 1/202.80 1.80 1.00 1/40 2.80 1.80 1.00 1/80 2.80 1.80 1.00 1/160 2.80 1.801.00 1/320 2.80 1.80 1.00 1/640 2.80 1.80 1.00 Citrate 2.80 1.80 1.00Buffer

TABLE 19 The residual virus titers and log reductions of infectiousA/Panama/2007/99 (H3N2) virus after the 1-minute termination time pointat different dilutions. 1 Minute Incubation A/Panama Residual InfluenzaVirus log Virus Titer titer reductions Dilution (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) 1/10 4.80 3.80 1.00 1/20 4.80 3.80 1.00 1/404.80 4.80 0.00 1/80 4.80 4.30 0.50 1/160 4.80 4.80 0.00 1/320 4.80 4.800.00 1/640 4.80 4.80 0.00 Citrate 4.80 0.00 4.80 Buffer

TABLE 20 The residual virus titers and log reductions of infectiousA/Panama/2007/99 (H3N2) virus after the 2-minute termination time pointat different dilutions. 2 Minute Incubation A/Panama Residual InfluenzaVirus log Virus Titer titer reductions Dilution (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) 1/10 4.80 3.80 1.00 1/20 4.80 4.30 0.50 1/404.80 4.80 0.00 1/80 4.80 4.30 0.50 1/160 4.80 4.80 0.00 1/320 4.80 4.800.00 1/640 4.80 4.80 0.00 Citrate 4.80 2.30 2.50 Buffer

TABLE 21 The residual virus titers and log reductions of infectiousA/Panama/2007/99 (H3N2) virus after the 5-minute termination time pointat different dilutions. 5 Minute Incubation A/Panama Residual InfluenzaVirus log Virus titer titer reductions Dilution (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) 1/10 4.80 3.80 1.00 1/20 4.80 4.30 0.50 1/404.80 4.80 0.00 1/80 4.80 4.80 0.00 1/160 4.80 4.80 0.00 1/320 4.80 4.800.00 1/640 4.80 4.80 0.00 Citrate 4.80 2.80 2.00 Buffer

TABLE 22 The residual virus titers and log reductions of infectiousB/Guangdong/120/00 virus after the 1-minute termination time point atdifferent dilutions. 1 Minute Incubation B/Guangdong Residual InfluenzaVirus log Virus Titer titer reductions Dilution (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) 1/10 3.30 1.30 2.00 1/20 3.30 1.80 1.50 1/403.30 2.80 0.50 1/80 3.30 2.80 0.50 1/160 3.30 2.80 0.50 1/320 3.30 2.800.50 1/640 3.30 2.80 0.50 Citrate 3.30 0.00 3.30 Buffer

TABLE 23 The residual virus titers and log reductions of infectiousB/Guangdong/120/00 virus after the 2-minute termination time point atdifferent dilutions. 2 Minute Incubation B/Guangdong Residual InfluenzaVirus log Virus Titer titer reductions Dilution (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) 1/10 3.30 1.80 1.50 1/20 3.30 1.80 1.50 1/403.30 2.80 0.50 1/80 3.30 2.80 0.50 1/160 3.30 2.80 0.50 1/320 3.30 2.800.50 1/640 3.30 2.80 0.50 Citrate 3.30 0.00 3.30 Buffer

TABLE 24 The residual virus titers and log reductions of infectiousB/Guangdong/120/00 virus after the 5-minute termination time point atdifferent dilutions. 5 Minute Incubation B/Guangdong Residual InfluenzaVirus log Virus titer titer reductions Dilution (−log₁₀ TCID₅₀) (−log₁₀TCID₅₀) (−log₁₀ TCID₅₀) 1/10 3.30 1.80 1.50 1/20 3.30 1.80 1.50 1/403.30 2.80 0.50 1/80 3.30 2.80 0.50 1/160 3.30 2.80 0.50 1/320 3.30 3.300.00 1/640 3.30 1.80 1.50 Citrate 3.30 0.00 3.30 Buffer

In the viricidal assay, a known titer of Influenza virus was used as thevirus control; this control underwent the same procedures as the testcompound, QR-435. The Influenza titer on all plates was consistent witha virus control titer greater than 2.5 (−log₁₀ TCID₅₀).

EXAMPLE 8 In Vivo Testing of Antimicrobial Spray

The ferret is an established animal model for the study of influenzainfection (Boyd and Beeson, 1975; Chen et al., Induction and Relief ofnasal Congestion in Ferrets Infected with Influenza, Int. J. Exp.Pathol. 76: 55-64, 1995; Scheiblauer et al., Pathogenicity of InfluenzaA/Seal/Mass/1/80 Virus Mutants for Mammalian Species, Arch. Virol. 140:341-8, 1995; Sweet and Smith, Pathogenicity of Influenza Virus,Microbiol. Rev. 44: 303-30, 1980; Toms et al., The relation of Pyrexiaand Nasal Inflammatory Response to Virus Levels in Nasal Washings ofFerrets Infected with Influenza Viruses of differing Virulence, Br. J.Exp. Pathol., 58: 444-58, 1997; Webster et al., Protection of FerretsAgainst Influenza Challenge with a DNA Vaccine to haemagglutinin,Vaccine 12: 1495-8, 1994). The ferret model has previously been used todetermine the efficacy of influenza vaccines (Fenton et al., 1981;Webster et al., 1994). Transmission studies that utilized the ferretanimal model have not only demonstrated donor to recipient spread ofinfluenza virus, but also the effects of mutations on the virulence ofthe virus (Herlocher et al., Ferrets as a Transmission Model forInfluenza: Sequence changes on the HA1 of type A (H3N2) Virus, J. ofInfect. Diseases 184: 542-46, 2001; Herlocher et al., Influenza viruscarrying an R292K Mutation in the Neuraminidase Gene is not Transmittedin Ferrets, Antimicrobial Res. 54: 99-111, 2002).

Five minutes before being infected with an influenza virus of the strainA/Sydney/5/97 (H3N2), four groups of six naïve ferrets receivedintranasal doses of experimental or control compositions. The negativecontrol group received a phosphate buffer solution (PBS) placebo. Thepositive control group was treated with Tamiflu™ (oseltamivir phosphate,available from Roche Laboratories of Nutley, N.J.). One experimentalgroup was treated with the nasal spray of Example 6, and the other wastreated with a similar nasal spray that did not include green teaextract. After the initial challenge, the ferrets were dosed with theirassigned composition twice a day.

The ferrets in the PBS treated control group exhibited all the symptomstypical of ferrets infected with influenza A, including weight loss,fever, increased inflammatory cell counts, and virus shedding on thefirst day after infection. The ferrets in the Tamiflu™ treated controlgroup experienced no weight loss, no virus shedding, a reduction ininflammatory cell count rise, and no febrile illness.

Both the test formulation of Example 6 and the similar nasal spray thatdid not include green tea extract provided a low-level intermediaryreduction in inflammatory cell count, prevented development of a febrileillness, and delayed virus shedding in a manner that may indicate virussuppression. Ferrets treated with nasal spray according to Example 6,however, also showed some lessening of weight loss. Ferrets treated withnasal spray according to Example 6 were more active than ferrets treatedwith the Tamiflu™.

EXAMPLE 9 In Vivo Transmissivity and Prophylaxis Testing

The properties of a nasal spray, QR-435, were tested. The formulationfor QR-435 was as follows:

QR-435 Formulation Oleoresin Turmeric 0.0308 weight percent Aquaresin ®Ginger 0.0326 weight percent Horseradish Oil #58 0.00300 weight percentGreen Tea PE 030725 0.0220 weight percent Glycerin 2.3368 weight percentDeionized Water 97.5749 weight percent

The study explored the effectiveness of the intranasal application byspray to prevent viral transmission to animals living in close proximityto an infected animal when the infected animal is treated and theprovision of prophylaxis to uninfected animals.

Fifty ferrets (albino or Fitch, from Highgate Farms, Lincolnshire, GB)approximately 6-8 months old, 700-1200 g in weight were divided into 6groups after electronic tagging, as shown in Table 25. Health scoreswere measured from day 0 to day 6 and the appropriate animal was placedunder anesthesia for intranasal infection, with 0.25 ml per nostril,with Influenza A/Sydney/5/97 [H3N2] allantoic stock. TABLE 25 TreatmentGroup Distribution Assignment Group Number of Ferrets Treatment 1 5Placebo-Transmission 2 5 Placebo-Prophylaxis 3 10 QR435-Prophylaxis 4 10QR435-Transmission 5 10 Tamiflu ™-Prophylaxis 6 10Tamiflu ™-TransmissionGroups 3 and 4 in the protocol are shown as group 3 above, groups 4 and5 are shown as group 4 above, groups 7 and 8 are shown as group 5 aboveand groups 9 and 10 are shown as group 6 above. The groups were pairedand subdivided into groups of 5 for animal welfare reasons only.

TABLE 26 Infection status of the donor animals Nasal Systemic VirusFerret ID # Group symptoms symptoms Seroconversion shedding 037284841Placebo-Transmission Y Y Y N 034537339 Placebo-Prophylaxis Y Y Y N034543003 QR435 Prophylaxis Y N Y N 036534054 QR435 Prophylaxis Y Y Y N034540555 QR435 Transmission Y Y No sample Low 036788799 QR435Transmission Y N No sample Low 036803821 Tamiflu ™ Prophylaxis N Y Y LowXXXC Tamiflu ™ Prophylaxis Y Y Y N XXXD Tamiflu ™ Transmission Y Y Y N036800314 Tamiflu ™ Transmission Y Y Y LowTransmission Study

The object of the transmission study was to determine whether the testcompound could prevent a donor ferret from transmitting influenza virusto non-infected recipient ferrets in the same pen. One ferret per groupwas inoculated with virus, and approximately 5 minutes afterinoculation, the remaining uninoculated animals were administered eitherthe test or the control compound. The volume of test compound or controlwas 0.14 ml per nostril for a total of 0.28 ml per dosing time. Theinoculated animal was isolated for 24 hours then re-introduced to theappropriate group on day 1. The inoculated animal was treated twicedaily with the test compound or appropriate control. The remainingferrets were not treated throughout the duration of the experiment. Ondays 0 through 6 all ferrets were observed for clinical signs, weightloss and fever. Intranasal wash collection was performed on day 6, thevolume of wash recovered measured and the weight of the nasal wash notedin the laboratory notebook. Viral titer of the recovered nasal washeswas determined using MDCK cells.

Tables 27-31 show the results of the transmission experiment. Table 27shows the percentage of animals with nasal symptoms of influenza. TABLE27 Percentage of animals with nasal symptoms of influenza (donor animalsexcluded) Day GROUP 1 2 3 4 5 6 Placebo 0% 0% 0% 100% 75%   50%Tamiflu ™ 0% 12.5%   37.5  50%  0% 12.5% QR435 0% 0% 0%  0% 25% 37.5%

Table 28 shows the percentage of animals with reduced physical activity.TABLE 28 Percentage of animals with reduced physical activity (donoranimals excluded) Day GROUP 1 2 3 4 5 6 Placebo 0% 0% 0% 100%  0% 0%Tamiflu ™ 0% 0% 0% 0% 0% 12.5%   QR435 0% 12.5%   0% 0% 12.5%   0%

The various criteria for influenza illness were considered and used todetermine the percentage of animals showing symptoms. When nasalsymptoms were considered (Table 27), by day 4, 100% ({fraction (4/4)})of the animals in the placebo group had symptoms of influenza, whereas,by contrast on day 4 only 50% ({fraction (4/8)}) of animals had symptomsin the Tamiflu™ treated group. None of the animals in the QR435treatment group had symptoms. By day 6 however 37.5% of the animals inthe QR435 treatment group had nasal symptoms. It is unclear as towhether these were caused by influenza infection or irritation from thenasal spray.

When systemic signs of influenza such as reduced activity areconsidered, by day 4, all ({fraction (4/4)}) animals in the placebogroup had symptoms, however only a single animal (12.5%) showed signs ofillness on any day post infection in the active treatment groups.

Table 29 shows laboratory confirmed influenza and seroconversion by thetreatment group. Each animal was bled before challenge and on day 24,post infection so that influenza infection could be confirmed byserology. Seroconversion was defined as a four-fold rise in anti HAIantibodies against A/Sydney/5/97 (H3N2). In Table 29 it is shown that100% of animals in both the placebo treatment group and the Tamiflu™treatment group seroconverted. However, only 57% ({fraction (4/7)}) ofthose animals tested seroconverted in the QR435 treatment group. Due toequipment failure one serum sample could not be tested. TABLE 29Laboratory confirmed influenza; Seroconversion by treatment group # ofanimals Percentage of animals seroconverted seroconverted Placebo 4/4100 QR435 4/7* 57 Tamiflu ™ 8/8 100*one animal was culled early and no sample taken

Table 30 shows the mean virus shedding from the nasal mucosa on day 6post infection. Nasal washes were performed on day 6 post infection andthe samples titrated on MDCK cells for influenza virus. Table 30 shows asubstantial difference in the number of animals shedding virus betweenthe two active treatment groups. Only a single animal in the QR435treatment group shed virus compared to all animals in the Tamiflu™treatment group. Unexpectedly, only a single animal in the placebo group(25%) shed virus. TABLE 30 Laboratory confirmed influenza; Virusshedding from the nasal mucosa on day 6 post infection # of animalsPercentage of shedding virus animals shedding virus GROUP on day 6 onday 6 Placebo 1/4  25% QR435 1/7*  14 Tamiflu ™ 8/8 100*one animal was culled early and no sample taken

The mean virus shedding by treatment group was calculated and thedifferences compared by ANOVA. The result was found to be statisticallysignificant (p=0.02). T-tests were performed using the Bonferronicorrection for multiple testing and the Tamiflu™ treated group was foundto shed significantly more virus than both the placebo treatment group(p=0.021) and the QR435 treatment group (p=0.04).

Table 31 is the mean maximum health score by treatment group. The meanand Standard Deviation of the maximum health score was calculated asdescribed above in the prophylaxis study results. The results weresignificant (p=0.02). Individual groups were compared as described aboveand both treatment groups have significantly lower maximum health scoresthan the placebo group (QR435 p=0.006 and Tamiflu™ p=0.003). TABLE 31Mean Maximum Health Score by Treatment Group. Group Mean N Std.Deviation Placebo 1.0000 4 .00000 QR435 .8750 8 .35355 Tamiflu ™ .7500 8.70711 Total 1.0500 20 .68633

An AUC-like measure comprising the sum of health scores post infectionwas calculated for each animal; treatment group's means and StandardDeviations were calculated and compared ANVOA.

Table 32 shows the results and the difference between treatment groupswas highly significant (p=0.002). Individual groups were compared usingt-tests and the Bonferroni correction for multiple testing, bothtreatment groups had significantly lower total health scores than theplacebo group (QR435 p=0.06 and Tamiflu™ p=0.03). TABLE 32 Mean TotalHealth Score by Treatment Group Group Mean N Std. Deviation Placebo4.2000 4 .95743 QR435 .3750 8 .64087 Tamiflu ™ .5000 8 .83452 Total1.5000 20 1.53125

The mean maximum weight change post infection was similarly calculatedand compared by ANOVA. The results were not significant (p=0.90). Bothtreatment groups had significantly less weight loss than the placebogroup (QR435 p=0.055 and Tamiflu™ p=0.019). The AUC like measurecomparing the sum of the weight changes post infection was alsocalculated for each animal and compared by ANOVA. The difference betweentreatment groups was not significant (p=0.64).

Prophylaxis Study

The object of the prophylaxis study was to determine whether treatmentof uninfected recipient ferrets with the test compound could preventviral infection from an infected donor. One ferret per group wasinoculated with virus, and approximately 5 minutes after inoculation,the remaining uninoculated animals were administered either the test orthe control compound. The inoculated animal was isolated for 24 hoursthen re-introduced to the appropriate group on day 1. The inoculatedanimal was not treated with the test compound or appropriate control.The remaining ferrets were treated with the test compound or appropriatecontrol twice daily. The volume of test compound or control was 0.14 mlper nostril for a total of 0.28 ml per dosing time. On days 0 through 6all ferrets were observed for clinical signs, weight loss and fever.Intranasal wash collection was performed on day 6, the volume of washrecovered measured and the weight of the nasal wash noted in thelaboratory notebook. Viral titers of the recovered nasal washes weredetermined using MDCK cells. A bleed at 24 days was taken for controlassays.

Each donor animal was successfully infected. Due to the parameters ofthe studies it was important to demonstrate that each donor animal wassuccessfully infected, that each donor animal demonstrated clinicalsigns of infection and seroconversion. However, viral shedding was notdetected in the nasal washings. It is not surprising that there waslittle or no viral shedding in the donor animals as viral shedding oftenceases prior to day 6, the day the washing were taken.

Tables 33-38 show the results from the prophylaxis experiment. Table 33shows the percentage of animals that had nasal symptoms of influenza.Table 34 shows the percentage animals that had reduced physical activityduring the testing period. TABLE 33 Percentage of animals with nasalsymptoms of influenza (donor animals excluded) by treatment group DayGROUP 1 2 3 4 5 6 Placebo 0 0 0 0 75% 75% Tamiflu ™ 0 0 25% 0  0% 12.5QR435 0 0 0 0 25% 25%

TABLE 34 Percentage of animals with reduced physical activity (donoranimals excluded) Day GROUP 1 2 3 4 5 6 Placebo 0 0 100% 100% 0 0Tamiflu ™ 0 0 0 0 0 0 QR435 0 0 0 0 0 0Prophylaxis Study Results

In the prophylaxis study, 75% of the placebo group (¾) had nasalsymptoms of influenza infection by day 5, whereas 25% of the Tamiflu™group had nasal symptoms by day 3 and 25% of the QR435 group had nasalsymptoms by day 5. When the percentage of animals showing reducedphysical activity is considered the results are more dramatic in that100% of the placebo group showed reduced physical activity and none ofthe animals from either of the Tamiflu™ or the QR435 group showed anysigns of reduced activity. Table 35 shows the mean maximum health scoreby treatment group. The mean and standard deviation of the maximumhealth score for each animal was calculated by treatment group thencompared by ANOVA. The results approached significance (p=0.087). TABLE35 Mean Maximum Health Score by Treatment Group Group Mean N Std.Deviation Placebo 1.0000 4 .00000 QR435 .3750 8 .51755 Tamiflu ™ .3750 8.51755 Total .5000 20 .51299

An AUC-like measure comprising the sum of health scores post infectionwas calculated for each animal; treatment group's means and StandardDeviations were calculated and compared ANVOA. Table 35 shows theresults and the difference between treatment groups was highlysignificant (p<0.0005). Individual groups were compared using t-testsand the Bonferroni correction for multiple testing, both treatmentgroups had highly significantly lower total health scores than theplacebo group. TABLE 36 Mean Total Health Score by Treatment Group GroupMean N Std. Deviation Placebo 3.5000 4 1.00000 QR435 .3750 8 .51755Tamiflu ™ .5000 8 .75593 Total 1.5000 20 1.43178

Each animal was bled before challenge and on day 24, post infection, sothat influenza infection could be confirmed by serology. Seroconversionis a four-fold increase in HAI antibodies against the inoculationstrain. TABLE 37 Laboratory confirmed influenza; Seroconversion bytreatment group # of animals Percentage of animals seroconvertedseroconverted Placebo 2/2* 100% QR435 0/8  0% Tamiflu ™ 8/8 100%

Table 38 shows laboratory confirmed influenza and the percentage ofanimals virus shedding on day 6 post infection of the treatment group.Nasal washes were performed on day 6 post infection and the samplestitrated on MDCK cells for influenza virus. Table 38 shows that there isa substantial difference in the number of animals shedding virus betweenthe two active treatment groups, i.e., for the QR435 treated group noneof the animals shed virus compared to 75% in the placebo treated groupand 87.5% in the Tamiflu™ treated group. TABLE 38 Laboratory confirmedinfluenza; Virus shedding on day 6 post infection by treatment group #of animals shed- Percentage of animals ding virus on day 6 sheddingvirus on day 6 Placebo 3/4 75% QR435 0/8  0% Tamiflu ™ 7/8 87.5%  

The mean virus shedding by treatment group was calculated and thedifferences compared by ANOVA. The result was found to be statisticallysignificant (p=0.001). T-tests were performed using the Bonferronicorrection for multiple testing and the QR435 treated group was found toshed significantly less virus than both the placebo treatment group(p=0.004) and the Tamiflu™ treatment group (p=0.002).

The mean and standard deviation of the maximum weight change for eachanimal was calculated by treatment group and then compared by ANOVA. Theresults were significant (p=0.018). Individual groups were comparedusing t-tests and the Bonferroni correction for multiple comparisons.Both treatment groups had significantly less (or very close to) weightloss than the placebo treatment group (QR435 p=0.055 and Tamiflu™p=0.019).

An AUC-like measure comprising the sum of the weight changes postinfection was calculated for each animal; treatment group means andStandard Deviations were calculated and compared by ANOVA. Thedifference between treatment groups was not significant (p=0.461).

The mean maximum weight change post infection was similarly calculatedand compared by ANOVA. The results were significant (p=0.018). Bothtreatment groups had significantly less weight loss than the placebogroup (QR435 p=0.055 and Tamiflu™ p=0.019). The AUC like measurecomparing the sum of the weight changes post infection was alsocalculated for each animal and compared by ANOVA. The difference betweentreatment groups was not significant (p=0.461).

Conclusion

The positive treatment group that was treated with Tamiflu™ (oseltamivirphosphate, administered at 5 mg/kg twice daily) was generally protectedfrom the symptoms of influenza in both the transmission and prophylaxisexperiments. On the other hand, these animals were infected as shown byboth viral shedding and the seroconversion of the recipient animals. Incontrast, the test compound QR-435 was 100% effective in preventinginfection and partially effective in preventing transmission. Thus, theimportant benefit of the compound is its ability to prevent infection,particularly the suppression of the shedding of viral particles whichwould reduce the spread of disease.

EXAMPLE 10 In Vitro Comparison of the Virucidal and Virustatic ActivityAgainst the SARS Virus

Two compositions QR439 and QR439(a) were tested in vitro for virucidaland virustatic activity against the SARS virus. QR439 was formulated asfollows: (1) Aquaresin ® ginger 0.6849% by weight (2) Oleoresin turmeric0.6466% by weight (3) Green Tea PE 0.4619% by weight (4) Glycerin49.1039% by weight (5) Deionized water 49.1039% by weight QR439(a) wasformulated as follows: (1) Aquaresin ® ginger 0.6840% by weight (2)Oleoresin turmeric 0.6466% by weight (3) Green Tea PE 0.4619% by weight(4) Horseradish oil 0.063192% by weight (5) Glycerin 49.0723% by weight(6) Deionized water 49.0723% by weight

In the test, the positive control compounds were: 1% Triton-X 100 (inartificial saliva [0.1% NaHCO₃, 18% KH₂PO₄, 0.1% gastric mucin, pHadjusted to 6.0-6.5]) for the virucidal assay and Ribavirin (200 μ/ml)in 1% DMSO (in PBS) for the virustatic assay.

The negative control compound was Artificial Saliva (0.1% NaHCO₃, 18%KH₂PO₄, 0.1% gastric mucin, pH adjusted to 6.0-6.5) for the virucidalassay only. Urbani SARS virus was used at a stock titer of 10⁵TCID₅₀/ml. The test compounds, QR439 and QR439(a) were diluted inSterets Normasol (Seton Prebbles Ltd) to produce the following dilutionsfor the virucidal assay: 1/10, 1/20, 1/40, 1/80, 1/160, 1/320, and1/640.

Virucidal Assay Method

Residual virus titers were titrated on C1008 (a clone of Vero 76) cellsfor viral growth in 96-well plates. The results were analyzed for CPE(cytopathic effect) and the virus titer determined using the Karbermethod.

The following steps were conducted in performing the virucidal assay.360 μl of the test compound was mixed with 40 μl virus. The mixture wasagitated and then incubated for 30 seconds, 1, 2, 4 or 8 minutes. Thereaction was terminated at a specific time by adding 3.6 ml of infectionmedia (DMEM, 2 mM L-glutamine, HEPES, penicillin-streptomycin).Termination is caused by the dilution effect of the addition of theinfection media because both the virus and test compounds are dilutedten-fold in this step. Residual virus infectivity was titrated on C1008cells at 10 fold dilutions across the 96-well plate. The C1008 cellswere incubated for 7-10 days at 37° C. The results were scored for CPEand virus titer calculated. The cells were examined for CPE on Days 3,4, and 5 post-infection.

Cytotoxicity Assay Method

A cytotoxicity assay on C1008 cells was carried out on all threecompounds to determine which dilutions to use for the virustatic assay.200 μl of each test compound, and control compound, was added to thefirst wells (in duplicate) of a 96-well plate and then titrated acrossthe plate following a 2-fold dilution series. The plate was incubated at35° C., 5% CO₂. The cells were observed for CPE after 3 days incubation.

Virustatic Assay Method

The dilutions that were used for the virustatic assay are as follows:QR439 and QR439(a) at the following dilutions: 1/200, 1/300, 1/400,1/500, 1/600. Infection media (DMEM, 2 mM L-glutamine, HEPES,penicillin-streptomycin) was used to dilute each test compound, asappropriate. 100 μl of each diluted test compound, and the positivecontrol, were added to wells of C1008 cells (in duplicate) and thenincubated for 30 minutes at 35° C., 5% CO₂. After the incubation period,10 μl of Urbani SARS virus (at a stock titer of 10⁵ TCID₅₀/ml) was addedto each well of the test compounds and positive control. The plate wasincubated at 35° C., 5% CO₂ for 3-5 days before observing the cells forCPE. A crystal violet assay, which measured the OD (optical density) ofthe cells, was performed on the plate after the CPE was recorded. The ODvalues were used to determine the TC₅₀ and EC₅₀ of the test compounds.

Results

The results of the cytotoxicity assay performed on the test compoundsare shown in Table 39 shown below. Each undiluted compound was added induplicate to wells in the first column of the plate and diluted acrossthe plate in a 2-fold dilution series. The concentration within therange of the 1/8 and 1/16 dilutions is equivalent to the concentrationof the test compounds that were added to the plates in the virucidalassay. This is because the compounds were diluted 1/10 when thetermination media was added. TABLE 39 Cytotoxicity Assay Test 2-FoldDilution Series Compound UD 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512QR439 + + + + + + + + − − QR439(a) + + + + + + + + − − Cells Only − − −− − − − − − −+ both wells exhibited toxicity− both wells exhibit non-toxicity+/1 one well exhibits toxicity, the other well exhibits non-toxicity

Table 39 demonstrates that both test compounds are toxic to the cells atthe 1/8 dilution (which is equivalent to the dilution used in thevirucidal assay for the undiluted compounds). In the virucidal andvirustatic assays, a dilution of greater than 1/128 was needed toeliminate toxicity.

The CPE observations (Table 40) show that there was no virustaticactivity detected for any of the three compounds tested. TABLE 40 CPEObservations QR439 QR439(a) Ribovirin Cells only Virus ControlDilutions* of A + + T − + Test B + + T − + Compounds C + + T − + D + + T− + E + + T − + F ND ND T − + 10-Fold Dilutions Undiluted 10 10³ 10⁴ 10⁵Undiluted Virus + + +/− − −+ Virus infection in both wells− No viral infections in both wells+/− Viral infection in one well no infection in the other wellT Toxicity in both wellsND Virustatic assay not done at a sixth serial dilution*Dilutions used for QR439 and QR439(a) are A = 1/200, B = 1/300, C =1/400, D = 1/500, E = 1/600

The results of the virucidal assay performed on the test compounds QR439and QR439(a) are represented in Table 41 and Table 42 respectively. TheTables indicate the reduction in viral titer after 1/10 dilution of thetest compounds, which is equivalent to a 1/10 dilution of the undilutedvirus. TABLE 41 Log reduction in Viral Titer after contact with TestCompound QR439 Log Reduction in Viral Titer (−log₁₀TCID₅₀/ml) VirusInitial Test Compound Contact Times (minutes) Control Dilution 0.5 1 2 48 Titer^(a) Undiluted T T T T T 2.5 1/10 ≧1.0 ≧1.0 ≧1.0 ≧1.0 ≧1.0 2.51/20 ≧1.0 ≧1.0 ≧1.0 ≧1.0 ≧1.0 2.5 1/40 2.5* 2.5* 2.5* 2.5* 2.5* 3.0 1/802.0* 2.0* 2.0* 2.0* 2.0* 2.5 1/160 2.5* 2.5* 2.0 2.5* 2.5* 3.0 1/320 0.01.5 1.0 1.5 2.5* 3.0 1/1/640 0.0 0.5 1.5 0.5 1.0 3.0a −log₁₀TCID₅₀/ml*No virus recovery. However using the Karber Calculation method (takinginto account the initial 1/10 dilution) a titer of 0.5 TCID₅₀/ml isobtained

TABLE 42 Log reduction in Viral Titer after contact with Test CompoundQR439(a) Log Reduction in Viral Titer (−log₁₀TCID₅₀/ml) Virus InitialTest Compound Contact Times (minutes) Control Dilution 0.5 1 2 4 8Titer^(a) Undiluted T T T T T 2.5 1/10 ≧1.0 ≧1.0 ≧1.0 ≧1.0 ≧1.0 2.5 1/202.0* 2.0* 2.0* ≧1.0 ≧1.0 2.5 1/40 2.0* 2.0* 2.0* 2.0* 2.0* 3.0 1/80 2.02.0 2.5* 2.5* 2.5* 2.5 1/160 2.5 3.0* 3.0* 3.0* 3.0* 3.0 1/320 0.0 0.50.0 0.5 1.0* 1.5 1/1/640 0.5 0.5 1.5 0.5 0.0 2.5a −log₁₀TCID₅₀/ml*No virus recovery. However using the Karber Calculation method (takinginto account the initial 1/10 dilution) a titer of 0.5 TCID₅₀/ml isobtained

The results show that for QR439, there is a significant reduction invirucidal activity from the 1/40 to 1/640 dilutions at all contact timepoints, with the exception of 1/320 (30 seconds) and 1/640 (30 seconds,1 minute, and 4 minutes). The results show that for QR439(a) there is asignificant reduction in virucidal activity from the 1/20 to 1/60dilutions at all contact time points with the exception of 1/20 (4 and 8minutes).

EXAMPLE 11 Efficacy of Antimicrobial Nasal Spray on H3N2 InfluenzaInfection

Forty ferrets were infected with influenza A/Sydney/5/97 [H3N2] andtreated with QR435, as employed in Example 9 above, or control compoundsdaily for 4 days. Clinical signs were measured alongside weights, rectaltemperatures, cell counts, and virus shedding. The virus inoculumA/Sydney/5/97 was virulent and signs of illness were observed, with theplacebo treated (PBS) ferrets exhibiting distinct symptoms of influenzaA infection. The positive control (Tamiflu™) was effective at reducingweight loss, fever, and clinical symptoms. No reduction of illness wasnoted in the QR435 administered groups.

The test substance was a nasal spray containing 1.8% active compoundwith the formula as noted in example 9 above. The positive controlsubstance was Tamiflu™ (Osletamivir phosphate) administered at 5 mg/kgtwice daily. Phosphate buffered saline (as a nasal spray) was also beused as a control and administered intranasal four times a day.

The challenge virus was A/Sydney/5/97 [H3N2] was from the Retroscreenvirus repository. The ferrets were successfully infected with influenzaand exhibited the classical signs of the associated disease in the PBStreatments group (negative control). The Tamiflu™ treatment group(positive control) showed reduction in the severity of the disease withstatistically significant (or almost significant) reductions in, virusshedding, weight loss, and illness scores.

Neither of the two test compound treatment groups (QR435 twice daily andQR435 four times daily) demonstrated reduced illness for treatmentpurposes. However, as noted in example 9 above, QR435 has proveneffective for anti-transmissivity and prophylactic uses.

EXAMPLE 12

In an effort to reduce the irritant properties of the nasal spray, yetretain the anti-transmissivity properties previously demonstrated, theefficacy of varying horseradish concentrations was tested in the ferretmodel. The QR-435 full horseradish ALS sialorrhea spray formulation wasas follows:

QR-435 Formulation Oleoresin Turmeric 0.0308 weight percent Aquaresin ®Ginger 0.0326 weight percent Horseradish Oil #58 0.00300 weight percentGreen Tea PE 030725 0.0220 weight percent Glycerin 2.3368 weight percentDeionized Water 97.5749 weight percentIn the 50% horseradish test formulation, the horseradish oil was reducedto 0.0015% by weight and the glycerin and deionized water increased by0.000075% by weight. Similarly, in the 25% horseradish study, thehorseradish oil was reduced to 0.000075 and the glycerin and deionizedwater increased by 0.001125% by weight.

Table 43 shows the distribution of the various ferret groups and the pentreatment protocol. Each group was subdivided into two pens strictly forlogistical reasons. Each group consisted of four recipient animals andone donor animal. TABLE 43 Group Distribution Group Number of FerretsTreatment 1a 5 Placebo (PBS) QDS 1b 5 Placebo (PBS) QDS 2a 5 QR435 100%Horseradish QDS 2b 5 QR435 100% Horseradish QDS 3a 5 QR435 50%Horseradish QDS 3b 5 QR435 50% Horseradish QDS 4a 5 QR435 25%Horseradish QDS 4b 5 QR435 25% Horseradish QDS 5a 5 Tamiflu ™ 5 mg/kgBDS 5b 5 Tamiflu ™ 5 mg/kg BDSQDS = 4 times dosing per day intranasallyBDS = 2 times dosing per day orally

The animals were electronically tagged using programmable injectabletransponders for animal identification and body temperature monitoring.Blood samples were taken from superficial veins and the terminal bleedwas taken by cardiac puncture.

Donor ferrets where infected intranasally as follows: one animal fromthe group of five was removed and infected with 0.5 ml of virus (0.25 mlper nostril) while the animal was under anesthetic. The donor animalswere not treated with the test material. Recipient ferrets were treatedaccording to the treatment group assignment as outlined in Table 43 andstudy schedule in Table 44. Nasal wash collection was performed and thevolume of nasal wash recovered was measured and weighed to determine thevolume of nasal wash recovery.

The HI assay was performed upon the sample against homologous virus todetermine the seronegativity of the ferrets as well as determining theextent of seroconversion, if any, following infection. Health scores,body weight and temperature were recorded. Total cell counts on thenasal washing samples were determined using trypan blue in order toevaluate the inflammatory cell response recovered from the nasalepithelium. Virus shedding from the nasal washes were performed usingMDCK cells. TABLE 44 Study Schedule Day 0 1 2 3 4 5 6 16 ElectronicTagging X Anesthesia X X Temperature X X X X X X X Body Weight X X X X XX X Health score X X X X X X X Infection of Donor X Isolation of Donor XIntegration of Donor X Nasal wash X Serum for HAI X X Test ArticleDosing X X X X X X X Recipient

Table 45 shows the clinical signs of the donor animals after exposure tothe influenza virus. As can be seen from the table, each donor animalwas successfully infected with influenza virus. TABLE 45 Donor ClinicalSigns % Weight Loss Fever Sero- Pen* (Peak to Trough) >1SD, >2SD,or >3SD conversion 1a 0.80 >3SD Y 1b 2.31 >3SD Y 2a 2.88 >3SD Y 2b0.91 >3SD Y 3a 1.90 >2SD Y 3b 1.43 >2SD Y 4a 2.76 >2SD Y 4b 1.15 >3SD Y5a 2.49 >1SD Y 5b 0.93 >2SD Y*See Table 43 for Treatment Protocol

Influenza related illness in the recipient ferrets was determined usingthe following parameters: weight loss, fever, virus shedding,seroconversion, clinical nasal and activity signs, nasal cell counts.The control groups (PBS treated groups 1a and 1b) exhibited typicalsigns of influenza illness that included weight loss, fever,seroconversion, and the presence of some clinical signs of illness(including activity and nasal signs). The Tamiflu™ treatment groups didnot show any overt clinical signs and did not seroconvert, therefore theTamiflu™ treatment was confirmed as being effective at reducinginfluenza illness. Comparison of the percentage weight loss betweengroups suggests that there is a difference in illness. The weight losswas calculated by finding the peak weight of each ferret followed byidentifying the subsequent trough. The difference in weight between peakand trough was calculated as a percentage of the baseline weight astaken on day 0. When the groups were compared for weight loss bytreatment, several treatments exhibited significant reductions inillness. The results from Table 46 indicate that PBS treated group wassignificantly different from all other treatments. (PBS vs. 100%horseradish p=0.008; PBS vs. 50% horseradish p=0.022; PBS vs. 25%horseradish p=0.005; PBS vs. Tamiflu™ p=0.003).

Thus, the test compound was as effective as the positive control(Tamiflu™) at preventing weight loss in animals infected with influenzavia the natural transmission route, from another ferret. TABLE 46AResults Comparison Dependent Variable: Weight Change separated into pensStd. 95% Confidence Interval Group Number Mean Error Lower Bound UpperBound PBS −2.860 .405 −3.686 −2.034 100% horseradish −1.231 .405 −2.057−.405 50% horseradish −1.478 .405 −2.304 −.651 25% horseradish −1.141.405 −1.967 −.315 Tamiflu ™ −.980 .405 −1.806 −.154

TABLE 46B Pairwise Comparison Dependent Variable: Weight Changeseparated into pens Mean (I) Group Difference Std. 95% ConfidenceInterval Number (J) Group Number (I−J) Error Sig.^(a) Lower Bound UpperBound PBS 100% Horseradish −1.629 .572 .008 −2.797 −.460 50% horseradish−1.382 .572 .022 −2.551 −.214 25% horseradish −1.719 .572 .005 −2.887−.550 Tamiflu ™ −1.880 .572 .003 −3.048 −.712 100% PBS 1.629 .572 .008.460 2.797 horseradish 50% horseradish 2.46 .572 .670 −.922 1.415 25%horseradish −9.000E−02 .572 .876 −1.258 1.078 Tamiflu ™ −.251 .572 .664−1.420 .917 50% PBS 1.382 .572 .022 .214 2.551 horseradish 100%Horseradish −.246 .572 .670 −1.415 .922 25% horseradish −.336 .572 .561−1.505 .832 Tamiflu ™ −.497 .572 .391 −1.666 .671 25% PBS 1.719 .572.005 .550 2.887 horseradish 100% Horseradish −9.000E−02 .572 .876 −1.0781.258 50% horseradish .336 .572 .561 −1.666 1.505 Tamiflu ™ −.161 .572.780 −1.330 1.007 Tamiflu PBS −1.880 .572 .003 .712 3.048 100%Horseradish .251 .572 .664 −.917 1.420 50% horseradish .497 .572 .391−.671 1.666 25% horseradish .161 .572 .780 −1.007 1.330a. Adjustments for multiple comparisons: least significant difference(equivalent to no adjustments)

Individual ferrets that showed temperature rises greater than 3 SD(Standard Deviations) from the previous temperature measurement weredeemed to have a strong febrile illness. For each group the SD wascalculated from each group's mean temperature on Day 1. Due to the useof anesthesia on Day 0, and possible subsequent modifications of ferrettemperature, Day 1 temperatures were deemed more appropriate to be usedto generate the baseline mean and SD. Table 47 is a summary of fever asdefined as a 3 SD rise from the previous temperature measurement. TABLE47 Results Comparison No. of Ferrets per No. of ferrets per Pen pen withFever Group group with Fever Number* (>3SD rise) Number (>3SD rise) 1a 21 4 1b 2 2a 1 2 1 2b 0 3a 0 3 1 3b 1 4a 3 4 5 4b 2 5a 0 5 1 5b 1*See Table 43 for Pen Treatment Protocol

TABLE 48 Mild Febrile Illness No. of Ferrets per No. of ferrets per Penpen with Fever Group group with Fever Number* (>2SD rise) Number (>2SDrise) 1a 3 1 7 1b 4 2a 2 2 4 2b 2 3a 1 3 2 3b 1 4a 3 4 5 4b 2 5a 2 5 45b 2*See Table 43 for Pen Treatment Protocol

Using X² analysis of the fever as defined by a 3SD rise from theprevious temperature measurement the following was determined:

-   -   PBS treatment vs. 50% horseradish p=0.106 (Table 49)    -   PBS vs. 10% horseradish p=0.106 (Table 50)    -   PBS vs. Tamiflu™ p=0.106 (Table 51)

Table 48 summarizes the number of ferrets with a clear temperature riseof more than 2SD from the previous temperature measurement, which weredeemed to have a mild febrile illness. TABLE 49 X² analysis of thenumber of ferrets with fever between PBS and 50% horseradish treatment(>3SD rise in temperature from baseline) Group Number 100% PBShorseradish Total Fever => 3SD No Count 4 7 11 rise from Expected count5.5 5.5 11.0 baseline % within Group 50.0% 87.5% 68.8% No. yes Count 4 15 Expected count 2.5 2.5 5.0 % within Group 50.0% 12.5% 31.3% No. TotalCount 8 8 16 Expected count 8.0 8.0 16.0 % within Group  100%  100% 100% No. Chi-Squared Tests Asymp. Sig. Exact Sig. Exact Sig. Value df(2-sided) (2-sided) (1-sided) Pearson Chi-Square  2.618^(b) 1 .106Continuity  1.164 1 .281 Correction^(a) Likelihood Ration  2.756 1 .097Fisher's Exact Test .282 .141 Linear-by-Linear  2.455 1 .117 AssociationN of Valid Cases 16^(a)Computed only for 2 × 2 table^(b)2 cells (50.0%) have expected count less than 5. The maximumexpected count is 2.50.

TABLE 50 X² analysis of the number of ferrets with fever between PBS and100% horseradish treatment (>3SD rise in temperature from baseline)Group Number 50% PBS horseradish Total Fever => 3SD No Count 4 7 11 risefrom Expected count 5.5 5.5 11.0 baseline % within Group 50.0% 87.5%68.8% No. yes Count 4 1 5 Expected count 2.5 2.5 5.0 % within Group50.0% 12.5% 31.3% No. Total Count 8 8 16 Expected count 8.0 8.0 16.0 %within Group  100%  100%  100% No. Chi-Squared Tests Asymp. Sig. ExactSig. Exact Sig. Value df (2-sided) (2-sided) (1-sided) PearsonChi-Square  2.618^(b) 1 .106 Continuity  1.164 1 .281 Correction^(a)Likelihood Ration  2.756 1 .097 Fisher's Exact Test .282 .141Linear-by-Linear  2.455 1 .117 Association N of Valid Cases 16^(a)Computed only for 2 × 2 table^(b)2 cells (50.0%) have expected count less than 5. The maximumexpected count is 2.50.

TABLE 51 X² analysis of the number of ferrets with fever between PBS andTamiflu ™ treatment (>3SD rise in temperature from baseline) GroupNumber 50% PBS horseradish Total Fever => 3SD No Count 4 7 11 rise fromExpected count 5.5 5.5 11.0 baseline % within Group 50.0% 87.5% 68.8%No. yes Count 4 1 5 Expected count 2.5 2.5 5.0 % within Group 50.0%12.5% 31.3% No. Total Count 8 8 16 Expected count 8.0 8.0 16.0 % withinGroup  100%  100%  100% No. Chi-Squared Tests Asymp. Sig. Exact Sig.Exact Sig. Value df (2-sided) (2-sided) (1-sided) Pearson Chi-Square 2.618^(b) 1 .106 Continuity  1.164 1 .281 Correction^(a) LikelihoodRation  2.756 1 .097 Fisher's Exact Test .282 .141 Linear-by-Linear 2.455 1 .117 Association N of Valid Cases 16^(a)Computed only for 2 × 2 table^(b)2 cells (50.0%) have expected count less than 5. The maximumexpected count is 2.50.

Using X² analysis of the fever as defined by a 2SD rise from theprevious temperature measurement the following was determined:

-   -   PBS treatment vs. 50% horseradish p=0.012 (Table 52)    -   PBS vs. 10% horseradish p=0.106 (Table 53)

PBS vs. Tamiflu™ p=0.106 (Table 54) TABLE 52 X² analysis of the numberof ferrets with fever between PBS and 50% horseradish treatment (>2SDrise in temperature from baseline) Group Number 50% PBS horseradishTotal Fever => 2SD No Count 1 6 7 rise from Expected count 3.5 3.5 7.0baseline % within Group 12.5% 75% 68.8% No. yes Count 7 2 9 Expectedcount 4.5 4.5 9.0 % within Group 87.5% 25% 56.3% No. Total Count 8 8 16Expected count 8.0 8.0 16.0 % within Group  100%  100%  100% No.Chi-Squared Tests Asymp. Sig. Exact Sig. Exact Sig. Value df (2-sided)(2-sided) (1-sided) Pearson Chi-Square  6.349^(b) 1 .012 Continuity 4.063 1 .044 Correction^(a) Likelihood Ration 26.904 1 .009 Fisher'sExact Test .041 .020 Linear-by-Linear  5.952 1 .015 Association N ofValid Cases 16^(a)Computed only for 2 × 2 table^(b)4 cells (100.0%) have expected count less than 5. The maximumexpected count is 3.50.

TABLE 53 X² analysis of the number of ferrets with fever between PBS and100% horseradish treatment (>2SD rise in temperature from baseline)Group Number 100% PBS horseradish Total Fever => 2SD No Count 1 6 5 risefrom Expected count 2.5 2.5 5.0 baseline % within Group 12.5% 50% 31.3%No. yes Count 7 4 11 Expected count 5.5 5.5 11.0 % within Group 87.5%50% 68.8% No. Total Count 8 8 16 Expected count 8.0 8.0 16.0 % withinGroup  100%  100%  100% No. Chi-Squared Tests Asymp. Sig. Exact Sig.Exact Sig. Value df (2-sided) (2-sided) (1-sided) Pearson Chi-Square 2.618^(b) 1 .106 Continuity  1.164 1 .281 Correction^(a) LikelihoodRation  2.756 1 .097 Fisher's Exact Test .282 .141 Linear-by-Linear 2.455 1 .117 Association N of Valid Cases 16^(a)Computed only for 2 × 2 table^(b)2 cells (50.0%) have expected count less than 5. The maximumexpected count is 2.50.

TABLE 54 X² analysis of the number of ferrets with fever between PBS andTamiflu ™ treatment (>2SD rise in temperature from baseline) GroupNumber PBS Tamiflu ™ Total Fever => 2SD No Count 1 4 5 rise fromExpected count 2.5 2.5 5.0 baseline % within Group 12.5% 50% 31.3% No.yes Count 7 4 11 Expected count 5.5 5.5 11.0 % within Group 87.5% 50%68.8% No. Total Count 8 8 16 Expected count 8.0 8.0 16.0 % within Group 100%  100%  100% No. Chi-Squared Tests Asymp. Sig. Exact Sig. ExactSig. Value df (2-sided) (2-sided) (1-sided) Pearson Chi-Square 2.618^(b) 1 .106 Continuity  1.164 1 .281 Correction^(a) LikelihoodRation  2.756 1 .097 Fisher's Exact Test .282 .141 Linear-by-Linear 2.455 1 .117 Association N of Valid Cases 16^(a)Computed only for 2 × 2 table^(b)2 cells (50.0%) have expected count less than 5. The maximumexpected count is 2.50.

Conclusions

While all test substance treated ferrets seroconverted, QR-435 with 100%and 50% horseradish components were found to either significantly ornearly significantly, reduce the systemic illness as measured by weightand temperature. However, the other clinical signs of illness were notabrogated by any treatment. The control groups displayed clinical signsof influenza illness. The PBS treated groups exhibit the typical signsof influenza infection: weight loss, fever, and seroconversion as wellas low clinical signs of illness, reduced activity and nasal signs. TheTamiflu™ treated groups show no significant weight loss, low clinicalsigns, virus shedding, reduced severity of fever and no seroconversion.

In the test treatment groups, the severity of illness was defined byfever and weight loss. All treatment dosages significantly reduced theseverity of weight loss when compared to the PBS group. When fever wasdefined as a 2SD rise in temperature, the 50% horseradish treatmentgroup showed significant reductions in the incidence of fever. The 100%horseradish treatment group also abrogated fever and was found to benearly as significant. When fever was defined as a 3 SD rise intemperature, the 100% and 50% horseradish treatment groups had nearlysignificant reductions in the incidence of fever, but the 25%horseradish treatment did not abrogate fever. Thus, the 100% and the 50%horseradish treatments were found to reduce systemic signs to a greaterdegree than the 25% horseradish treatment. This suggests a strong dosedependency requiring 50% or greater horseradish to abrogate both feverand weight loss. Treatment with 50% or greater horseradish may reducethe titer of virus transmitted from the donor to the recipient therebyreducing the severity of disease symptoms. It is also possible thattreatment with 50% or greater horseradish abrogated illness by acting ina therapeutic manner.

EXAMPLE 13

The first documented case of a human infection with an avian strain wasthe 1997 Hong Kong H5N1 infection of 18 persons resulting in 6 deaths. Amore recent outbreak has occurred in Southeast Asia resulting innumerous deaths as shown in Table 55. TABLE 55 Confirmed Human H5N1(Avian) Influenza Cases in SE Asia (WHO 2004) Country Total No. of casesNo. of Deaths Vietnam 22 15 Thailand 12 8 Total 34 23

This study investigated the efficacy of the formulations given in Tables56-57 below, against a human isolate of an avian strain influenza virusA/Vietnam/1194/04, H5N1. The negative control was DMEM-DMSO 1% while thepositive control in the virustatic assay was amatadine at 0.37 μM. Thepositive control for the virucidal assay was 1% Tween-20/20% ETOH/PBS(final concentration). In addition, cell only and virus only positivecontrols where used. The cell only control used maintenance media only.For the virus only control, the test procedures were identical exceptcell maintenance media was used instead of the test compounds. Tables56-57 display the formulations that were tested. TABLE 56 Formula IIngredient Weight % Oleoresin Tumeric, ex. Kalsec 0.6466 AquaresinGinger, ex. Kalsec. 0.6840 Horseradish - Horseradish oil, ex. Kalsec0.06312 Green Tea PE - 90% Phytoway 0.4619 Green Tea PE 030725. HuizhenS09160304 Glycerin 49.0723 Water 49.0723 TOTAL 100.0002

TABLE 57 Formula II Ingredient Weight % Tumeric Extract in VegetableOil. 0.6466 Ex. Kalsec Oleoresin Ginger. 0.6840 Ex. Kalsec.Horseradish - Horseradish oil, 0.06312 ex. Kalsec Green Tea PE - 90%0.4619 Phytoway Green Tea PE 030725. Huizhen S09160304 Glycerin 36.9077Water 36.9077 Neobee M-5 Medium Chain 12.2850 Triglyceride BASF T-MazPolysorbate 80K 12.0441 TOTAL 100.0001

The end point of the virucidal assay was determined by visualobservation of the cytopathic effects (CPE) and the residual viral titerwas determined by the Karber method. Reduction of the virus titerexposed to the test compounds was determined by comparison to the virusonly control. Antiviral activity is a reduction of 1−log₁₀ TCID₅₀/ml(See, Oxford, J. S., et al., “Sodium deoxycholate exerts a directdestructive effect on HIV and Influenza viruses in-vitro and inhibitsretrovirus-induced pathology in an animal model.” Antiv. Chem.Chemother., 5(3): 176-181 (1994)). The end point of the virustatic assaywas also determined by visual CPE scores. The antiviral activity wasrepresented by the presence or absence of infection.

Virustatic Assay

In the virustatic assay both formulations I-II were diluted 1/10 (10¹),1/100 (10²) and 1/1000 (10³). Additionally, undiluted formulations I-II(10⁰) were tested. In the virustatic assay, Mardin-Darby canine kidney(MDCK) cells were seeded into 96-well plates (100 μl/well) and incubatedfor 2 days at 37° C. with 5% CO₂. After 2 days, the cells were washedtwo times with 100 μl/well PBS then exposed to 100 μl/well infectionmedia. A 1/10³ dilution of neat viral stock was added to the cells andallowed to attach for 1 to 2 hours under standard incubator conditions(i.e. 37° C. with 5% CO₂). Each test formulation (diluted and undiluted)was then added to the infected wells at 50 μl/well. The plates wereincubated for 3 days under standard incubator conditions. CPEobservation and crystal violet staining determined the end point of theassay. Additionally, a hemagglutination assay (HA) was performedfollowing a 2-fold dilution series.

Under crystal violet staining, some virustatic activity was observedwhen formulation I was used undiluted and for the 1/10¹ dilution. Theeffective concentration, EC₅₀ was calculated to be 1/10^(1.88) (i.e. adilution between 1/10¹ and 1/10²).

In this assay, CPE was observed in all test wells for all dilutions offormulation II. Under crystal violet staining, no virustatic activitywas observed with formulation II.

Table 58 displays the HA assay data from the virustatic assay. TABLE 58Virustatic Assay Activity HA Titer (HAU) Dilution (10^(X)) Formulation INew Formulation II 0 64  <2* −1 32  <2* −2 256 128 −3 256 256*The HAU (HA Units) value recorded here is likely to be due to thetoxicity of the compound rather than the antiviral activity.

The HA titer for amatadine ranged from 16 to 32 HAU indicating littleactivity against the A/Vietnam/1194/04 strain.

Virucidal Assay

In the virucidal assay both Formulation I and II were diluted 1/10 and1/80. In the virucidal assay, Mardin-Darby canine kidney (MDCK) cellswere seeded into 96-well plates (100 μl/well) and incubated for 2 daysat 37° C. with 5% CO₂. After 2 days, the cells were washed two timeswith 100 μl/well PBS then exposed to 100 μl/well infection media. A1/1000 dilution of neat viral stock (40 μl/well) was added each testcompound (360 μl) and left to incubate at room temperature for either 30seconds or 5 minutes. The reaction was terminated by the addition of theinfection media (3.6 ml). Termination of the reaction was caused by the1:10 dilution. The termination mixture was added in duplicate (111 μl)to the first row of the 96 well plates and titrated across the platefollowing a 10-fold dilution series. The plates were incubated for 3days under standard incubator conditions and CPE scored. Ahemagglutionation assay (HA) was performed following a 2-fold dilutionseries.

Table 59 displays the HA data from the virucidal assay usingformulation. TABLE 59 Virucidal Activity of Formulation I Formulation I(HAU) 1/10 1/80 Time (minutes) Dilution (10^(X)) 0.5 5 0.5 5  0 <2 <2 64<2 −1 <2 <2 <2 <2 −2 <2 <2 <2 <2 −3 <2 <2 <2 <2 −4 <2 <2 <2 <2 Cellcontrol <2 <2 <2 <2 Virus control 64 64 64 32The data indicates that there is virucidal activity for formulation Iafter a 5 minute incubation and for the 1/10 dilution only after a 30second incubation.

Table 60 displays HA data from the virucidal assay using formulation II.TABLE 60 Virucidal Activity of Formulation II Formulation II (HAU) 1/101/80 Time (minutes) Dilution (10^(X)) 0.5 5 0.5 5  0 256 256 32 256 −1<2 <2 16 32 −2 <2 <2 <2 <2 −3 <2 <2 <2 <2 −4 <2 <2 <2 <2 Cell control <2<2 <2 <2 Virus control 32 64 64 64Table 60 shows that there is some detectible virucidal activity withformulation II but reduced quantitatively when compared to that observedfor formulation I. The positive control for both virucidal assays was 1%Tween-20/20% ETOH/PBS. The positive control had a <2 HAU consistently onall plates thus demonstrating good antiviral activity against theA/Vietnam/1194/04 strain.

Conclusion

Formulation I had detectable antiviral activity when tested in avirustatic assay. The activity was classified as moderate withapproximately a 10-fold reduction in virus replication. However, somenonspecific changes were observed in the cell monolayer when incubatedwith the QR formulations, hence the precise contribution of theanticellular versus antiviral effects could not be quantified. Certainnovel antivirals have been designated to exert viral inhibiting activityvia an effect on cellular function. It is possible that the differentcomponents of the QR mixture are exerting different anticellular andantiviral effects.

Virucidal activity was detected in formulation II but formulation II hada lower efficacy than formulation I. One possible future study could beto investigate the contribution of each constituent of the completecompound.

To date NI (neuramidase inhibitor) blockers have been shown to inhibitthe avian influenza strain H5N1, whereas amantadine has been shown to berelatively inactive against avian H5N1 strains.

It is to be understood, however, that even though numerouscharacteristics and advantages of the present invention have been setforth in the foregoing description, they are illustrative only. Changesmay be made in carrying out the methods and to the compositions of theinvention above set forth above without departing from the spirit andscope of the invention. It is intended that all matter contained in theabove description shall be interpreted as illustrative and not in alimiting sense. The scope of this invention is to be determined from theclaims appended hereto.

1. A method for reducing the transmissivity of a microbe, comprising thestep of administering to a mammal or a bird that has been exposed to anillness caused by a microbe or has contracted an illness caused by amicrobe, a safe and effective amount of composition comprising: a firstingredient obtainable from ginger; a second ingredient obtainable fromgreen tea; an acceptable carrier; said amount being effective, whenadministered, to reduce an incidence of contracting said illness byanother mammal or bird exposed to said mammal or bird.
 2. The method ofclaim 1, wherein said illness is caused by a microbe selected from thegroup consisting of: a virus, a bacterium, a fungi and a yeast.
 3. Themethod of claim 2, wherein the microbe is a virus selected from thegroups consisting of herpes virus, HIV virus, AIDS virus, influenzavirus, H5N1 influenza virus, rhinovirus, the SARS virus, and respiratorysyncytial virus.
 4. The method of claim 1, wherein the composition isadministered in a form selected from a group consisting of a tablet, acapsule, a lozenge, a troche, a hard candy, a chewable composition, anda dental product.
 5. The method of claim 1, wherein the composition isadministered as a nasal spray or as a throat spray.
 6. The method ofclaim 1, wherein the first ingredient is selected from a groupconsisting of ginger powder extract, ginger fluid extract, gingerpowder, at least a part of a whole plant of ginger, a ginger tincture,one or more compounds contained in ginger, and mixtures thereof; and thesecond ingredient is selected from the group consisting of green teapowder, green tea powder extract, green tea fluid extract, at least apart of a whole plant of green tea, tinctures of green tea, one or morecompounds contained in green tea, and mixtures thereof.
 7. The method ofclaim 1, wherein the first ingredient comprises ginger root powder andthe second ingredient comprises green tea extract.
 8. The method ofclaim 1, wherein each gram of the composition contains about 1 mg toabout 20 mg of green tea extract, and about 1 mg to about 150 mg ofginger root powder.
 9. The method of claim 1, wherein the compositionfurther comprises a third ingredient obtainable from turmeric.
 10. Themethod of claim 9, wherein the ingredient obtainable from turmeric isselected from a group consisting of turmeric powder extract, turmericfluid extract, turmeric extract, one or more curcuminoid compounds, oneor more other compounds contained in turmeric, turmeric powder, at leasta part of a whole plant of turmeric, a turmeric tincture, and mixturesthereof.
 11. The method of claim 9, wherein the ingredient obtainablefrom turmeric comprises turmeric extract.
 12. The method of claim 9,wherein the composition contains about 1 mg to about 20 mg of turmericpowder extract.
 13. The method of claim 9, wherein the compositionfurther comprises a fourth ingredient obtainable from horseradish root.14. The method of claim 1, wherein the composition further comprises oneor more ingredients selected from the group consisting of ethanol,resveratrol, propylene glycol and glycerine.
 15. The method of claim 14,wherein the composition comprises ethanol.
 16. A method for theprophylactic use of a composition to reduce one or more of severity ofan illness, severity of symptoms of the illness, and incidence ofsymptoms of the illness, comprising the step of administering to amammal or a bird that has been, or will be, exposed to the illness, anamount of a composition comprising: a first ingredient obtainable fromginger; a second ingredient obtainable from green tea; an acceptablecarrier; said amount being effective, when administered, to reduce oneor more of the severity of the illness, the severity of symptoms of theillness, and the incidence of symptoms of the illness.
 17. The method ofclaim 16, wherein said illness is caused by a microbe from selected fromthe group consisting of: a virus, a bacterium, a fungi and a yeast. 18.The method of claim 17, wherein the virus is selected from the groupconsisting of herpes virus, HIV virus, AIDS virus, influenza virus, H5N1influenza virus, rhinovirus, the SARS virus, and respiratory syncytialvirus.
 19. The method of claim 16, wherein the composition isadministered in a form selected from a group consisting of a tablet, acapsule, a lozenge, a troche, a hard candy, a chewable composition, anda dental product.
 20. The method of claim 16, wherein the composition isadministered as a nasal spray or as a throat spray.
 21. The method ofclaim 16, wherein the first ingredient is selected from a groupconsisting of ginger powder extract, ginger fluid extract, gingerpowder, at least a part of a whole plant of ginger, a ginger tincture,one or more compounds contained in ginger, and mixtures thereof; and thesecond ingredient is selected from the group consisting of green teapowder, green tea powder extract, green tea fluid extract, at least apart of a whole plant of green tea, tinctures of green tea, one or morecompounds contained in green tea, and mixtures thereof.
 22. The methodof claim 16, wherein the first ingredient comprises ginger root powderand the second ingredient comprises green tea extract.
 23. The method ofclaim 16, wherein each gram of the composition contains about 1 mg toabout 20 mg of green tea extract, and about 5 mg to about 150 mg ofginger root powder.
 24. The method of claim 16, wherein the compositionfurther comprises a third ingredient obtainable from turmeric.
 25. Themethod of claim 24, wherein the ingredient obtainable from turmeric isselected from a group consisting of turmeric powder extract, turmericfluid extract, turmeric extract, one or more curcuminoid compounds, oneor more other compounds contained in turmeric, turmeric powder, at leasta part of a whole plant of turmeric, a turmeric tincture, and mixturesthereof.
 26. The method of claim 24, wherein the ingredient obtainablefrom turmeric comprises turmeric extract.
 27. The method of claim 24,wherein the composition contains about 1 mg to about 20 mg of turmericpowder extract.
 28. The method of claim 16, wherein the compositionfurther comprises a fourth ingredient obtainable from horseradish root.29. The method of claim 16, wherein the composition further comprisesone or more ingredients selected from the group consisting of ethanol,resveratrol, propylene glycol and glycerine.
 30. The method of claim 29,wherein the composition comprises ethanol.